Organization of higher-order oligomers of the serotonin₁(A) receptor explored utilizing homo-FRET in live cells

The serotonin_1A receptor is a representative member of the GPCR superfamily and serves as an important drug target. The possible role of GPCR oligomerization in receptor function is an active area of research. We monitored the oligomerization state of serotonin_1A receptors using homo-FRET and fluorescence lifetime measurements. Homo-FRET is estimated by a reduction in fluorescence anisotropy and provides a superior approach for exploring oligomerization. In addition, homo-FRET offers the possibility of detecting higher-order oligomers. On the basis of an observed increase in fluorescence anisotropy upon progressive photobleaching and analysis of the difference between the extrapolated anisotropy and the predicted anisotropy of an immobile monomer, we propose the presence of constitutive oligomers of the serotonin_1A receptor. To the best of our knowledge, these results constitute the first report of higher-order oligomers for the serotonin_1A receptor. We further show that cholesterol depletion and antagonist treatment result in a reduced population of higher-order oligomers. In contrast, agonist stimulation and destabilization of the actin cytoskeleton lead to an increased contribution from higher oligomers. These results provide novel insight into the oligomerization status of the serotonin_1A receptor that could enhance the ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.     

Metabolic depletion of sphingolipids enhances the mobility of the human serotonin1A receptor

Sphingolipids are essential components of eukaryotic cell membranes. We recently showed that the function of the serotonin_1A receptor is impaired upon metabolic depletion of sphingolipids using fumonisin B₁ (FB₁), a specific inhibitor of ceramide synthase. Serotonin_1A receptors belong to the family of G-protein coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral and developmental functions. Since function and dynamics of membrane receptors are often coupled, we monitored the lateral dynamics of the serotonin_1A receptor utilizing fluorescence recovery after photobleaching (FRAP) under these conditions. Our results show an increase in mobile fraction of the receptor upon sphingolipid depletion, while the diffusion coefficient of the receptor did not exhibit any significant change. These novel results constitute the first report on the effect of sphingolipid depletion on the mobility of the serotonin_1A receptor. Our results assume greater relevance in the broader context of the emerging role of receptor mobility in understanding cellular signaling.     

Membrane organization of the human serotonin1A receptor monitored by detergent insolubility using GFP fluorescence

Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT_1A receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin_1A receptor tagged to enhanced yellow fluorescent protein (5-HT_1AR-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT_1A receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT_1AR-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT_1AR-EYFP provide new insights into the membrane organization of the 5-HT_1A receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.     

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin1A receptor

The serotonin_1A (5-HT_1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT_1A receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT_1A receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT_1A receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT_1A receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT_1A receptor function.     

Effect of sphingomyelinase treatment on ligand binding activity of human serotonin1A receptors

The serotonin_1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. We have monitored the ligand binding of the human serotonin_1A receptor stably expressed in CHO cells (termed CHO-5-HT_1AR) following treatment with sphingomyelinase (SMase), an enzyme that specifically catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Our results show, for the first time, that the specific ligand binding activity of the serotonin_1A receptor in membranes isolated from CHO-5-HT_1AR cells is increased upon sphingomyelinase treatment. Saturation binding analysis reveals increase in binding affinity of the receptor under these conditions. This is accompanied by a reduction in membrane order, as monitored by fluorescence anisotropy of the membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in intact cells. These results represent the first report on the effect of sphingomyelinase treatment on the ligand binding activity of this important neurotransmitter receptor.     

Lack of adenosine A3 receptors causes defects in mouse peripheral blood parameters

The role of the adenosine A₃ receptor in hematopoiesis was studied using adenosine A₃ receptor knockout (A₃AR KO) mice. Hematological parameters of peripheral blood and femoral bone marrow of irradiated and untreated A₃AR KO mice and their wild-type (WT) counterparts were investigated. Irradiation of the mice served as a defined hematopoiesis-damaging means enabling us to evaluate contingent differences in the pattern of experimentally induced hematopoietic suppression between the A₃AR KO mice and WT mice. Defects were observed in the counts and/or functional parameters of blood cells in the A₃AR KO mice. These defects include statistically significantly lower values of blood neutrophil and monocyte counts, as well as those of mean erythrocyte volume, mean erythrocyte hemoglobin, blood platelet counts, mean platelet volume, and plateletcrit, and can be considered to bear evidence of the lack of a positive role played by the adenosine A₃ receptor in the hematopoietic system. Statistically significantly increased values of the bone marrow parameters studied in A₃AR KO mice (femoral bone marrow cellularity, granulocyte/macrophage progenitor cells, and erythrocyte progenitor cells) can probably be explained by compensatory mechanisms attempting to offset the disorders in the function of blood elements in these mice. The pattern of the radiation-induced hematopoietic suppression was very similar in A₃AR KO mice and their WT counterparts.     

Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M4 muscarinic receptors?

M₄ muscarinic acetylcholine receptor is a G protein-coupled receptor (GPCR) that has been associated with alcohol and cocaine abuse, Alzheimer''s disease, and schizophrenia which makes it an interesting drug target. For many GPCRs, the high-affinity fluorescence ligands have expanded the options for high-throughput screening of drug candidates and serve as useful tools in fundamental receptor research. Here, we explored two TAMRA-labelled fluorescence ligands, UR-MK342 and UR-CG072, for development of assays for studying ligand-binding properties to M₄ receptor. Using budded baculovirus particles as M₄ receptor preparation and fluorescence anisotropy method, we measured the affinities and binding kinetics of both fluorescence ligands. Using the fluorescence ligands as reporter probes, the binding affinities of unlabelled ligands could be determined. Based on these results, we took a step towards a more natural system and developed a method using live CHO-K1-hM₄R cells and automated fluorescence microscopy suitable for the routine determination of unlabelled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. Both image analysis methods were suitable for measuring fluorescence ligand saturation binding and kinetics as well as for screening binding affinities of unlabelled ligands.     

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala₁₂₂₇ to Ser₁₂₅₁), which contains a single Trp residue (W₁₂₄₆) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala₁₂₂₇ was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring (∼50% α-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp₁₂₄₆ to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including λ_max, lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.     

Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH)₂D₃, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.     

Functional selectivity of adenosine A1 receptor ligands?

The concept of functional selectivity offers great potential for the development of drugs that selectively activate a specific intracellular signaling pathway. During the last few years, it has become possible to systematically analyse compound libraries on G protein-coupled receptors (GPCRs) for this ‘biased’ form of signaling. We screened over 800 compounds targeting the class of adenosine A₁ receptors using a β-arrestin-mediated signaling assay in U2OS cells as a G protein-independent readout for GPCR activation. A selection of compounds was further analysed in a G protein-mediated GTPγS assay. Additionally, receptor affinity of these compounds was determined in a radioligand binding assay with the agonist [³H]CCPA. Of all compounds tested, only LUF5589 9 might be considered as functionally selective for the G protein-dependent pathway, particularly in view of a likely overestimation of β-arrestin signaling in the U2OS cells. Altogether, our study shows that functionally selective ligands for the adenosine A₁ receptor are rare, if existing at all. A thorough analysis of biased signaling on other GPCRs also reveals that only very few compounds can be considered functionally selective. This might indicate that the concept of functional selectivity is less common than speculated.     

Hydrogen Peroxide Formation by Cells Treated with a Tumor Promoter

To determine whether oxidants capable of DNA modification are produced by cells treated with tumor promoters, we adapted a fluorometric method to our needs. HeLa cells were preincubated with 2‘,7‘-dichlorofluorescin diacetate (DCFdAc), treated with various agents, sonicated. centrifuged and fluorescence of the oxidized product (DCF) was determined in supernatants. When cells were exposed to H₂O₂ in the presence of azide (catalase inhibitor) or o-phenanthroline (a lipophilic Fe chelator), an increase in fluorescence was observed. These results show that some Fe ions were interacting with the H₂O₂ which entered the cells, thus decreasing its levels available for oxidation of the substrate and potentially increasing formation of OH, known DNA-damaging species. Glutathione (GSH). which is present in cells in substantial amounts, was found to reduce DCF whereas azide counteracted GSH-mediated reduction.

Treatment of HeLa cells with 12–0-tetradecanoyl-phorbol-13-acetate (TPA) in the presence of DCFdAc and azide resulted in dose-and time-dependent formation of DCF. Even when cells were sonicated prior to incubation with TPA, DCF was formed at levels proportional to the number of cells as well as dose of TPA. Flow cytometry of TPA-treated cells confirmed these findings.

These results demonstrate that tumor promoters can cause oxidative activation of HeLa cells, which produce active oxygen species, most likely H₂O₂, that ultimately contribute to the formation of oxidized bases such as 5-hydroxymethyl uracil in cellular DNA. They also show that this fluorometric method can be utilized for determination of cellular H₂O₂ formation at nM concentrations.     

CO2 and vitamin B12 interactions determine bioactive trace metal requirements of a subarctic Pacific diatom

Phytoplankton growth can be limited by numerous inorganic nutrients and organic growth factors. Using the subarctic diatom Attheya sp. in culture studies, we examined how the availability of vitamin B₁₂ and carbon dioxide partial pressure (pCO₂) influences growth rate, primary productivity, cellular iron (Fe), cobalt (Co), zinc (Zn) and cadmium (Cd) quotas, and the net use efficiencies (NUEs) of these bioactive trace metals (mol C fixed per mol cellular trace metal per day). Under B₁₂-replete conditions, cells grown at high pCO₂ had lower Fe, Zn and Cd quotas, and used those trace metals more efficiently in comparison with cells grown at low pCO₂. At high pCO₂, B₁₂-limited cells had ∼50% lower specific growth and carbon fixation rates, and used Fe ∼15-fold less efficiently, and Zn and Cd ∼3-fold less efficiently, in comparison with B₁₂-replete cells. The observed higher Fe, Zn and Cd NUE under high pCO₂/B₁₂-replete conditions are consistent with predicted downregulation of carbon-concentrating mechanisms. Co quotas of B₁₂-replete cells were ∼5- to 14-fold higher in comparison with B₁₂-limited cells, suggesting that >80% of cellular Co of B₁₂-limited cells was likely from B₁₂. Our results demonstrate that CO₂ and vitamin B₁₂ interactively influence growth, carbon fixation, trace metal requirements and trace metal NUE of this diatom. This suggests the need to consider complex feedback interactions between multiple environmental factors for this biogeochemically critical group of phytoplankton in the last glacial maximum as well as the current and future changing ocean.     

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin_1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin_1A receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin_1A receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin_1A receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.     

Nitrogen fixation and transfer in open ocean diatom–cyanobacterial symbioses

Many diatoms that inhabit low-nutrient waters of the open ocean live in close association with cyanobacteria. Some of these associations are believed to be mutualistic, where N₂-fixing cyanobacterial symbionts provide N for the diatoms. Rates of N₂ fixation by symbiotic cyanobacteria and the N transfer to their diatom partners were measured using a high-resolution nanometer scale secondary ion mass spectrometry approach in natural populations. Cell-specific rates of N₂ fixation (1.15–71.5 fmol N per cell h⁻¹) were similar amongst the symbioses and rapid transfer (within 30 min) of fixed N was also measured. Similar growth rates for the diatoms and their symbionts were determined and the symbiotic growth rates were higher than those estimated for free-living cells. The N₂ fixation rates estimated for Richelia and Calothrix symbionts were 171–420 times higher when the cells were symbiotic compared with the rates estimated for the cells living freely. When combined, the latter two results suggest that the diatom partners influence the growth and metabolism of their cyanobacterial symbionts. We estimated that Richelia fix 81–744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners. This study provides new information on the mechanisms controlling N input into the open ocean by symbiotic microorganisms, which are widespread and important for oceanic primary production. Further, this is the first demonstration of N transfer from an N₂ fixer to a unicellular partner. These symbioses are important models for molecular regulation and nutrient exchange in symbiotic systems.     

Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N₂) to ammonium (NH₄⁺), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N₂ fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N₂ fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N₂ fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N₂ fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO₂⁻ and PO₄³⁻ are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N₂ fixation may increase in the future.     

Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A2A receptors in striatal projection neurons

D₁- and D₂-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A₁-type receptors are located in both neuron classes, and adenosine A_2A-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca²⁺-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D₁-type receptors increase, while D₂-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, Ca_V1 Ca²⁺-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A₁- and A_2A-receptors have not been compared observing their actions on Ca²⁺-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca²⁺-currents by A₁-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A₁- and A_2A-receptors. We demonstrate that A₁-type receptors reduced Ca²⁺-currents in all SPNs tested. However, A_2A-type receptors enhanced Ca²⁺-currents only in half tested neurons. Intriguingly, to observe the actions of A_2A-type receptors, occupation of A₁-type receptors had to occur first. However, A₁-receptors decreased Ca_V2 Ca²⁺-currents, while A_2A-type receptors enhanced current through Ca_V1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.     

Effect of cholesterol content on the structural and dynamic membrane properties of DMPC/DSPC large unilamellar bilayers

In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3 mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5 °C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3 mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation time, as well as the fluorescence quenching of DPH by TEMPO, are at maxima, while the fluorescence intensity of dehydroergosterol and the lipid solubility in TritonX-100 are at minima. These results correlate well with the hypothesis of domain segregation in the DMPC/DSPC/Cholesterol LUV system. In this context, we postulate that at 33.3 mol% of Cho, the proportion of ordered domains reaches a maximum.     

Scintillation proximity assay (SPA) as a new approach to determine a ligand’s kinetic profile. A case in point for the adenosine A<Subscript>1</Subscript> receptor

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a “mix and measure” format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A₁ receptor (hA₁R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k _on and k _off) of unlabeled hA₁R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.     

Adenosine A<Subscript>1</Subscript> receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury

Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A₁ receptor deficiency (A₁R^?/?) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction size evaluated. Flow cytometry was performed on brain-infiltrating cells, and semi-automated analysis of flow cytometric data was applied. A₁R^?/? mice displayed larger infarctions (+33 %, p?<?0.05) and performed worse in beam walking tests (44 % more mistakes, p?<?0.05) than wild-type (WT) mice. Myeloid cell activation after injury was enhanced in A₁R^?/? versus WT brains. Activated B lymphocytes expressing IL-10 infiltrated the brain after HI in WT, but were less activated and did not increase in relative frequency in A₁R^?/?. Also, A₁R^?/? B lymphocytes expressed less IL-10 than their WT counterparts, the A₁R antagonist DPCPX decreased IL-10 expression whereas the A₁R agonist CPA increased it. CD4⁺ T lymphocytes including FoxP3⁺ T regulatory cells, were unaffected by genotype, whereas CD8⁺ T lymphocyte responses were smaller in A₁R^?/? mice. Using PCA to characterize the immune profile, we could discriminate the A₁R^?/? and WT genotypes as well as sham operated from HI-subjected animals. We conclude that A₁R signaling modulates IL-10 expression by immune cells, influences the activation of these cells in vivo, and affects outcome after HI.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.