Dopamine D2 receptor-mediated modulation of adenosine A2A receptor agonist binding within the A2AR/D2R oligomer framework

The molecular interaction between adenosine A_2A and dopamine D₂ receptors (A_2ARs and D₂Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine–dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson’s disease). While the effects of A_2AR challenge on D₂R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D₂R-mediated allosteric modulation of A_2AR binding when an A_2AR/D₂R oligomer is established. Thus, we synthesized fluorescent A_2AR agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A_2AR binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D₂R-mediated negative allosteric modulation on A_2AR agonist binding that was oligomer-formation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A_2AR agonist binding. Overall, the here-developed methods were found valid to explore the ability of drugs acting on D₂Rs to modulate A_2AR binding, thus serving to facilitate the preliminary selection of D₂R-like candidate drugs in the management of Parkinson’s disease.     

Reduction of airway anion secretion via CFTR in sphingomyelin pathway

The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (Isc) measurement revealed that isoproterenol (ISO, 0.1 microM)-induced anion secretion was prevented by pretreatment with SMase (0.3 U/ml, for 30 min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolinone (1-EBIO, a Ca2+-activated K+ channel opener)-induced Isc were unaffected. The effects of SMase were reproduced in responses to forskolin (20 microM) or 8-bromo-cAMP (2 mM). C2-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C2-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K+ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion.     

Involvement of intracellular and extracellular Ca2+ in tetramethylpyrazine-induced colonic anion secretion

In the present study we investigated the role of Ca(2+) in tetramethylpyrazine (TMP)-induced anion secretion in the human colonic epithelial cell line, Caco-2, using the short-circuit current (I(SC)) technique in conjunction with intracellular Ca(2+) measurements. The results showed that TMP-induced I(SC) response was significantly reduced by 58.8% and 38.3% after inhibiting Ca(2+) ATPase of endoplasmic reticulum (ER) with thapsigargin and mobilizing ER stored Ca(2+) release with ATP, respectively. Conversely, thapsigargin- and ATP-evoked I(SC) responses were also significantly reduced by pretreatment with TMP by 43.2% and 38.5%, respectively. Conversely, removal of extracellular Ca(2+), apical but not basolateral, or the presence of the Ca(2+) chelator (EGTA) significantly increased TMP-induced I(SC) by 47.1% and 37.8%, respectively. Similar to TMP, thapsigargin-induced current increase was also enhanced by chelating extracellular Ca(2+) or in Ca(2+) free solution; however, removal of extracellular Ca(2+) did not significantly affect 3-isobutyl-1-methylxanthine (IBMX)- and forskolin-induced transepithelial current. Measurement of the intracellular concentration of free Ca(2+) ([Ca(2+)](i)) with fura-2/AM showed that TMP could induce an increase in [Ca(2+)](i) but pretreatment with TMP significantly reduced thapsigargin-evoked, but not ATP-induced, [Ca(2+)](i) increase. These results suggest that the effect of TMP on colonic anion secretion is partly mediated by TMP-increased [Ca(2+)](i) by acting on a target similar to thapsigargin. The observed inhibitory effect of extracellular Ca(2+) on Ca(2+)-dependent anion secretion represents a novel mechanism by which Ca(2+)-dependent regulation of epithelial electrolyte transport may be fine-tuned by extracellular Ca(2+) in the apical domain.     

Autocrine regulation of volume-sensitive anion channels in airway epithelial cells by adenosine

The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.     

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A₁, A_2A, A_2B and A₃ adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA₁ and hA_2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A₁/A_2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA₁ K_i?=?10.2?nM; hA_2A K_i?=?4.72?nM) and behaved as a potent A₁/A_2A antagonist/inverse agonist (hA₁ IC₅₀?=?13.4?nM; hA_2A IC₅₀?=?5.34?nM).     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

Adenosine A2A receptor activation supports an atheroprotective cholesterol balance in human macrophages and endothelial cells

The adenosine A_2A receptor (A_2AR) plays an important role in the regulation of inflammatory and immune responses. Our previous work has demonstrated that A_2AR agonists exhibit atheroprotective effects by increasing expression of reverse cholesterol transport proteins in cultured human macrophages. This study explores the impact of pharmacologic activation/inhibition and gene silencing of A_2AR on cholesterol homeostasis in both THP-1 human monocytes/macrophages and primary human aortic endothelial cells (HAEC).     

Cocaine produces D2R-mediated conformational changes in the adenosine A2AR-dopamine D2R heteromer

Adenosine A_2A receptors (A_2ARs) and dopamine D₂ receptors (D₂Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A_2ARs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A_2AR and D_2LR formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D₂R homodimers and in A_2AR-D₂R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET_max between A_2AR-D_2LR heterodimers and D_2LR homodimers, but not A_2AR homodimers, indicating its effect on D₂R. Cocaine was evaluated with regard to D₂R binding using a human D_2LR stable expressing CHO cell line and was found to produce an increase in the affinity of hD_2LR for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPγS. However, cocaine failed to modulate D₂R agonist-induced inhibition of cAMP in stable hD_2LR CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D_2LR, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D₂R homodimers and A_2AR-D₂R heterodimers.     

Nucleoside conjugates of quantum dots for characterization of G protein-coupled receptors: strategies for immobilizing A2A adenosine receptor agonists

Background  

Quantum dots (QDs) are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs).     

Indirect effects of adenosine triphosphate on chloride secretion in mammalian colon

Summary The effects of adenosine triphosphate (ATP) on shortcircuit current (SCC) in rat colonic epithelium are described. ATP caused a large increase in inward-going current and was considerably more potent in this respect than ADP. AMP or adenosine. The response to ATP was sided, there being only minor effects when the nucleotide was added to the apical side of the tissue. The effects of ATP were not modified by the cyclooxygenase inhibitor, indomethacin, eliminating eicosanoid formation as a mechanism. The effects of ATP were potentiated by theophylline and not blocked by ,-methylene ATP. The data are consistent with the effect being dependent on the activation of adenylate cyclase, but it has not been possible to classify the receptors intoP ₁ orP ₂ categories. Using inhibitors of NaCl cotransport (piretanide), carbonic anhydrase (acetazolamide), and chloride channels (diphenylamine-2-carboxylate), it was concluded that the SCC response to ATP was due to chloride secretion with, perhaps, a minor contribution from bicarbonate. Flux measurements with²²Na and³⁶Cl confirmed this view, there being approximate equivalence of chloride secretion with the SCC responses. Additionally, flux measurements revealed an inhibition of electroneutral NaCl absorption in response to ATP.The effects of ATP were antagonized by tetrodotoxin (TTX), greater than 50% inhibition being achieved with 10nm TTX. This result suggests that ATP does not act directly on receptors in the epithelial cells but rather on neuronal elements in the lamina propria. It will be necessary to re-examine other secretagogues for indirect effects of this kind and to search for the final effector neurotransmitter which evokes secretion.     

Evidence that histidine forms a coordination bond to the A0A and A0B chlorophylls and a second H-bond to the A1A and A1B phylloquinones in M688HPsaA and M668HPsaB variants of Synechocystis sp. PCC 6803

The axial ligands of the acceptor chlorophylls, A_0A and A_0B, in Photosystem I are the Met sulfur atoms of M688_PsaA and M668_PsaB. To determine the role of the Met, His variants were generated in Synechocystis sp. PCC 6803. Molecular dynamics simulations on M688H_PsaA show that there exist low energy conformations with the His coordinated to A_0A and possibly H-bonded to A_1A. Transient EPR studies on M688H_PsaA indicate a more symmetrical electron spin distribution in the A_1A phyllosemiquinone ring consistent with the presence of an H-bond to the C1 carbonyl. Ultrafast optical studies on the variants show that the 150 fs charge separation between P₇₀₀ and A₀ remains unaffected. Studies on the ns timescale show that 57% of the electrons are transferred from A_0A⁻ to A_1A in M688H_PsaA and 48% from A_0B⁻ to A_1B in M668H_PsaB; the remainder recombine with P₇₀₀⁺ with 1/e times of 25 ns and 37 ns, respectively. Those electrons that reach A_1A and A_1B in the branch carrying the mutation are not transferred to F_X, but recombine with P₇₀₀⁺ with 1/e times of ~ 15 μs and ~ 5 μs, respectively. Hence, the His is coordinated to A₀ in all populations, but in a second population, the His may be additionally H-bonded to A₁. Electron transfer from A₀ to A₁ occurs only in the latter, but the higher redox potentials of A₀ and A₁ as a result of the stronger coordination bond to A₀ and the proposed second H-bond to A₁ preclude electron transfer to the Fe/S clusters.     

Protein-cofactor interactions in bioenergetic complexes: The role of the A1A and A1B phylloquinones in Photosystem I

This review focuses on phylloquinone as an indispensable link between light-induced charge separation and subsequent charge stabilization in Photosystem I (PS I). Here, the role of the polypeptide in conferring the necessary kinetic and thermodynamic properties to phylloquinone so as to specify its functional role in PS I electron transfer is discussed. Photosynthetic electron transfer and the role of quinones in Type I and Type II reaction centers are introduced at the outset with particular emphasis on the determination of redox potentials of the cofactors. Currently used methodologies, particularly time-resolved optical spectroscopy and varieties of magnetic resonance spectroscopy that have become invaluable in uncovering the details of phylloquinone function are described in depth. Recent studies on the selective alteration of the protein environment and on the incorporation of foreign quinones either by chemical or genetic means are explored to assess how these studies have improved our understanding of protein-quinone interactions. Particular attention is paid to the function of the H-bond, methyl group and phytyl tail of the phylloquinone in interacting with the protein environment.     

Regulation of paracellular ion conductances by NaCl gradients in renal epithelial cells

In the present study, we clarified how the NaCl gradient across the epithelial cells regulates the paracellular ion conductance. Under isotonic conditions, the absorption-directed NaCl gradient elevated the paracellular conductances of Na(+) (G(Na)) and Cl(-) (G(Cl)), while the secretion-directed NaCl gradient diminished the G(Na) and G(Cl). We further investigated the paracellular ionic conductances of NMDG (G(NMDG)) and gluconate (G(gluconate)) by replacing Na(+) with NMDG or Cl(-) with gluconate. The G(NMDG) was lower than the G(Na) and the replacement of Na(+) with NMDG decreased the G(Cl). The G(gluconate) was lower than the G(Cl) and the replacement of Cl(-) with gluconate also decreased the G(Na). These observations suggest the interaction of cations and anions on paracellular ionic conductances; i.e., cations affect paracellular anion conductances and anions affect paracellular cation conductances.     

Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells

Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE₂ is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE₂, but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A₂-α, while facilitated AA mobilization and synthesis of PGE₂ within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA₂-α-driven enhanced AA mobilization and PGE₂ production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.     

Alterations in the distribution of glycoproteins in epithelial cells of murine colon after injection of tunicamycin

Summary Glycoproteins are associated with several structures of colonic absorptive cells of the mouse. These include the cell coat, Golgi apparatus and vesicles that transport the glycoproteins from the apparatus to the cell surface (Michaels and Leblond 1976). In many in vitro systems, the antibiotic tunicamycin inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins. In the present in vivo study, tunicamycin was injected into mice. The murine colonic epithelial cells were prepared routinely for electron microscopy and cytochemistry. Cells from the experimental and control animals were similar morphologically. However, staining by the periodic acid-chromic acid-silver methenamine technique, revealed differences in the distribution of glycoproteins. In animals that received the higher dosages of tunicamycin there was a substantial reduction in silver staining in both the Golgi apparatus and the vesicles of colonic epithelial cells compared to these structures in cells of identically treated control tissues, whereas the staining over the cell coat was not significantly altered. Possible explanations for the staining of the cell coat in the treated animals were provided in the text. This report demonstrates the feasibility of using tunicamycin in vivo and detection of the changes obtained by the silver methenamine method.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Antagonists of the human A2A receptor. Part 6: Further optimization of pyrimidine-4-carboxamides

Antagonists of the human A_2A receptor have been reported to have potential therapeutic benefit in the alleviation of the symptoms associated with neurodegenerative movement disorders such as Parkinson’s disease. As part of our efforts to discover potent and selective antagonists of this receptor, we herein describe the detailed optimization and structure–activity relationships of a series of pyrimidine-4-carboxamides. These optimized derivatives display desirable physiochemical and pharmacokinetic profiles, which have led to promising oral activity in clinically relevant models of Parkinson’s disease.     

Nuclear localization of the tight junction protein ZO-2 in epithelial cells

The tight junction constitutes the major barrier to solute and water flow through the paracellular space of epithelia and endothelia. It is formed by transmembrane proteins and submembranous molecules such as the MAGUKs ZOs. We have previously found that several MAGUKs, including those of the tight (ZO-1, ZO-2, and ZO-3) and septate junction (tamou and Dlg), contain one or two nuclear sorting signals located at their first PDZ and GK domains. Now we show that these proteins also contain a nuclear export signal and focus our study on the nuclear membrane shuttling of ZO-2. In sparse cultures this molecule concentrates at the nucleus in clusters, where it partially colocalizes with splicing factor SC35. Nuclear staining diminishes as the monolayer acquires confluence through a process sensitive to the nuclear export inhibitor leptomycin B. Nuclear localization can be induced by impairing cell-cell contacts, by mechanical injury. ZO-2 that shuttles from the cell periphery into the nucleus is not newly synthesized but originates from a preexistent pool. The movement of this protein is mediated by the actin cytoskeleton.