Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation

We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y(1) and P2Y(12) nucleotide receptors, 2-MeSADP, by blocking the beta-phosphate group with a 1-(3,4-dimethyloxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itself inactive at human P2Y(1) and P2Y(12) receptors expressed heterologously in 1321N1 astrocytoma cells or in washed human platelets, this derivative readily regenerates the parent agonist upon mild irradiation with long-wave UV light (360 nm). The functional effect of the regenerated agonist was demonstrated by a rise in intracellular calcium mediated by either P2Y(1) or P2Y(12) receptors in transfected cells. Washed human platelets exposed to a solution of MRS2703 were induced to aggregate upon UV irradiation. At 1.0 microM MRS2703, full aggregation was achieved within 1 min of irradiation. Thus, this caged nucleotide promises to be a useful probe for potent P2Y receptor activation with light-directed spatial and temporal control.     

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells

CHO cells stably transfected with adenosine receptors are widely utilized models for binding and functional studies. The effector coupling of human A3 adenosine receptors expressed in such a cellular model was characterized. Inhibition of adenylyl cyclase via a pertussis toxin-sensitive G protein was confirmed and exhibited a pharmacological profile in accordance with agonist binding data. The agonist potency was dependent on the assay system utilized to measure cyclase inhibition. Agonists were more potent in a cell-based assay than in experiments where cyclase inhibition was measured in a membrane preparation suggesting that receptor-effector coupling might be more efficient in intact cells. In addition to the modulation of cyclase activity, stimulation of A3 receptors elicited a Ca2+ response in CHO cells with agonist potencies corresponding to the values for the whole cell cAMP assay. The Ca2+ signal was completely eliminated by pertussis toxin treatment suggesting that it is mediated via betagamma release from a heterotrimeric G protein of the Gi/o family. These results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.     

Role of adenosine A3 receptors on CA1 hippocampal neurotransmission during oxygen-glucose deprivation episodes of different duration

The role of adenosine A3 receptors in synaptic transmission under severe (7 min) and shorter (2-5 min) ischemic conditions, obtained by oxygen and glucose deprivation (OGD), was investigated in rat hippocampal slices. The effects of selective A3 agonists or antagonists were examined on field excitatory postsynaptic potentials (fEPSPs) extracellularly recorded at the dendritic level of the CA1 pyramidal region. The novel, selective A3 antagonist LJ1251 ((2R,3R,4S)-2-(2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol, 0.1-10 nM) protected hippocampal slices from irreversible fEPSP depression induced by severe OGD and prevented or delayed the appearance of anoxic depolarization. Similar results were obtained when severe OGD was carried out with a long, receptor-desensitizing exposure to various selective A3 agonists: 5'-N-methylcarboxamidoadenosine derivatives Cl-IB-MECA (N6-(3-iodobenzyl)-2-chloro), VT72 (N6-methoxy-2-phenylethynyl), VT158 (N6-methoxy-2-phenylethynyl), VT160 (N6-methoxy-2-(2-pyridinyl)-ethynyl), and VT163 (N6-methoxy-2-p-acetylphenylethynyl) and AR132 (N6-methyl-2-phenylethynyladenosine). The selective A3 antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate, 100 nM) reduced fEPSP depression evoked by 2-min OGD and induced a faster recovery of fEPSP amplitude after 5-min OGD. Similar results were obtained for 2- or 5-min OGD applied in the presence of each of the A3 agonists tested. Shorter exposure to A3 agonists significantly delayed the recovery of fEPSP amplitude after 5-min OGD. This indicates that A3 receptors, stimulated by selective A3 agonists, undergo desensitization during OGD. It is inferred that CA1 hippocampal A3 receptors stimulated by adenosine released during brief ischemia (2 and 5 min) might exert A1-like protective effects on neurotransmission. Severe ischemia would transform the A3 receptor-mediated effects from protective to injurious.     

Downregulation of glutathione S-transferase M1 protein in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced mouse bladder carcinogenesis

Bladder cancer is highly recurrent following specific transurethral resection and intravesical chemotherapy, which has prompted continuing efforts to develop novel therapeutic agents and early-stage diagnostic tools. Specific changes in protein expression can provide a diagnostic marker. In our present study, we investigated changes in protein expression during urothelial carcinogenesis. The carcinogen BBN was used to induce mouse bladder tumor formation. Mouse bladder mucosa proteins were collected and analyzed by 2D electrophoresis from 6 to 20 weeks after commencing continuous BBN treatment. By histological examination, the connective layer of the submucosa showed gradual thickening and the number of submucosal capillaries gradually increased after BBN treatment. At 12-weeks after the start of BBN treatment, the urothelia became moderately dysplastic and tumors arose after 20-weeks of treatment. These induced bladder lesions included carcinoma in situ and connective tissue invasive cancer. In protein 2D analysis, the sequentially downregulated proteins from 6 to 20 weeks included GSTM1, L-lactate dehydrogenase B chain, keratin 8, keratin 18 and major urinary proteins 2 and 11/8. In contrast, the sequentially upregulated proteins identified were GSTO1, keratin 15 and myosin light polypeptide 6. Western blotting confirmed that GSTM1 and NQO-1 were decreased, while GSTO1 and Sp1 were increased, after BBN treatment. In human bladder cancer cells, 5-aza-2′-deoxycytidine increased the GSTM1 mRNA and protein expression. These data suggest that the downregulation of GSTM1 in the urothelia is a biomarker of bladder carcinogenesis and that this may be mediated by DNA CpG methylation.     

Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors

The thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of agonists and antagonists at cannabinoid CB₁ and CB₂ receptors were determined by means of affinity measurements at different temperatures and van’t Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB₁ and CB₂ receptors by inhibition assays of the binding of the cannabinoid receptor agonist [³H]-CP-55,940. van’t Hoff plots were linear for agonists and antagonists in the temperature range 0-30 °C. The thermodynamic parameters for CB₁ receptors fall in the ranges 17 ≤ ΔH° ≤ 59 kJ/mol and 213 ≤ ΔS° ≤ 361 kJ/mol for agonists and −52 ≤ ΔH° ≤ −26 kJ/mol and −12 ≤ ΔS° ≤ 38 kJ/mol for antagonists. The thermodynamic parameters for CB₂ receptors fall in the ranges 27 ≤ ΔH° ≤ 48 kJ/mol and 234 ≤ ΔS° ≤ 300 kJ/mol for agonists and −19 ≤ ΔH° ≤ −17 kJ/mol and 43 ≤ ΔS° ≤ 74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB₁ and CB₂ receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB₁ and CB₂ binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.     

Mood disorders: regulation by metabotropic glutamate receptors

Medicinal therapies for mood disorders neither fully serve the efficacy needs of patients nor are they free of side-effect issues. Although monoamine-based therapies are the primary current treatment approaches, both preclinical and clinical findings have implicated the excitatory neurotransmitter glutamate in the pathogenesis of major depressive disorders. The present commentary focuses on the metabotropic glutamate receptors and their relationship to mood disorders. Metabotropic glutamate (mGlu) receptors regulate glutamate transmission by altering the release of neurotransmitter and/or modulating the post-synaptic responses to glutamate. Convergent biochemical, pharmacological, behavioral, and clinical data will be reviewed that establish glutamatergic neurotransmission via mGlu receptors as a biologically relevant process in the regulation of mood and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. Specifically, compounds that antagonize mGlu2, mGlu3, and/or mGlu5 receptors (e.g. LY341495, MGS0039, MPEP, MTEP) exhibit biochemical effects indicative of antidepressant effects as well as in vivo activity in animal models predictive of antidepressant efficacy. Both preclinical and clinical data have previously been presented to define NMDA and AMPA receptors as important targets for the modulation of major depression. In the present review, we present a model suggesting how the interplay of glutamate at the mGlu and at the ionotropic AMPA and NMDA receptors might account for the antidepressant-like effects of glutamatergic- and monoaminergic-based drugs affecting mood in patients. The current data lead to the hypothesis that mGlu-based compounds and conventional antidepressants impact a network of interactive effects that converge upon a down regulation of NMDA receptor function and an enhancement in AMPA receptor signaling.     

Novel effect of carvedilol on Ca2+ movement in renal tubular cells

The effect of carvedilol on intracellular free Ca(2+) levels ([Ca(2+)](i)) has not been explored previously. This study was aimed to examine the effect of carvedilol on Ca(2+) handling in renal tubular cells. Madin-Darby canine kidney cells were used as a model for renal tubular cells and fura-2 was used as a fluorescent Ca(2+) probe. Carvedilol increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 5 microM. Extracellular Ca(2+) removal partly inhibited the [Ca(2+)](i) signals. Carvedilol-induced Ca(2+) influx was verified by measuring Mn(2+)-induced quench of fura-2 fluorescence. Carvedilol-induced store Ca(2+) release was reduced by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) but not with 5 microM ryanodine or 2 microM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Carvedilol (30 microM)-induced Ca(2+) release was not affected by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-l)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; 2 microM), but was potentiated by increasing cAMP levels or inhibiting protein kinase C. The carvedilol-induced Ca(2+) mobilization was not significantly sequestered by the endoplasmic reticulum or mitochondria. This study shows that carvedilol increased [Ca(2+)](i) in renal tubular cells by causing Ca(2+) release from the endoplasmic reticulum and other unknown stores in an inositol-1,4,5-trisphosphate-independent manner, and by inducing Ca(2+) influx. The Ca(2+) release was modulated by cAMP and protein kinase C.     

Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZ IMM125)-induced apoptosis in rat hepatocytes

The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3. Supplementing the cell cultures with the antioxidants, D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate, ascorbic acid, and the reducing agent, dithiothreitol, significantly inhibited the SDZ IMM125-mediated increase in chromatin condensation and fragmentation, and caspase-3 activity. D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate and dithiothreitol caused significant inhibition on SDZ IMM125-mediated cellular Ca(2+) uptake. The glutathione synthetase inhibitor, buthionine sulfoximine, increased SDZ IMM125-mediated caspase-3 action in parallel to chromatin condensation and fragmentation as well as Ca(2+) influx. Supplementation the culture medium with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid as well as omission of calcium in the medium reduced SDZ IMM125-induced apoptosis whereas the calcium supplementation of the culture medium elevated SDZ IMM125-induced apoptosis. Calcium antagonists inhibited SDZ IMM125-induced caspase-3 activation. Our data indicate that SDZ IMM125-mediated apoptosis in rat hepatocytes can be inhibited by antioxidants, and that the intracellular redox-state can act as a modulator of cytotoxicity and apoptosis. Further, the results suggest that SDZ IMM125-induced uptake of extracellular calcium is also a redox-sensitive process and that the increased intracellular calcium might directly cause apoptosis by increasing the caspase-3 activity as a central event in the cyclosporine-induced apoptotic mechanism.     

Modulation of dendritic release of dopamine by metabotropic glutamate receptors in rat substantia nigra

A superfusion system was used to study the effects of metabotropic glutamate receptor (mGluR) ligands upon the release of [(3)H]dopamine ([(3)H]DA) previously taken up by rat substantia nigra (SN) slices. trans-(+/-)-1-Amino-(1S,3R)-cyclopentane dicarboxylic acid (trans-ACPD; 100 and 600 microM), a group I and II mGluR agonist, evoked the release of [(3)H]DA from nigral slices. This last effect was reduced significantly by (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)-glycine (MCCG; 300 microM), an antagonist of group II mGluR, or by the addition of tetrodotoxin (D-APV; 1 microM) to the superfusion medium. D-(-)-2-Amino-5-phosphono-valeric acid (100 microM), an N-methyl-D-aspartate receptor antagonist, or the presence of Mg(2+) (1.2mM) in the superfusion medium did not modify trans-ACPD-induced [(3)H]DA release. In addition, a group II mGluR agonist such as (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG-IV; 100 microM) significantly induced the release of [(3)H]DA from nigral slices, whereas a group I mGluR agonist such as (RS)-3,5-dihydroxyphenylglycine (DHPG; 50 and 100 microM) did not modify the release of the [(3)H]-amine. Further experiments showed that the NMDA (100 microM)-evoked release of [(3)H]DA was decreased significantly by prior exposure of SN slices to trans-ACPD. Finally, partial denervation of the DA nigro-striatal pathway with 6-hydroxydopamine (6-OH-DA) increased trans-ACPD-induced release of [(3)H]DA, whereas it decreased trans-ACPD inhibitory effects on NMDA-evoked release of [(3)H]DA from nigral slices. The present results suggest that the dendritic release of DA in the SN is regulated by mGluR activation. Such nigral mGluR activation may produce opposite effects upon basal and NMDA-evoked release of DA in the SN. In addition, such mGluR-induced effects in the SN are modified in response to partial denervation of the DA nigro-striatal pathway.     

Behavioral effects of MK-801 in the rat

Several experiments were conducted to study the effects of the noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, on learning and memory in the rat. Rats displayed impaired performance on several sensorimotor tests and appeared grossly intoxicated when treated IP with 0.2 mg/kg MK-801, but not when treated with lower doses (0.05 or 0.1 mg/kg). Postacquisition performance on two spatial learning tasks involving working memory protocols (reinforced alternation and radial arm maze) was impaired by MK-801 at intoxicating doses (0.2 mg/kg) but not at lower doses (0.05 or 0.1 mg/kg). Using a position habit reversal task, we found that rats could learn to reverse a position habit while under the influence of a nonintoxicating dose of MK-801 (0.1 mg/kg), but when tested on the following day performed as if they did not recall what they had learned. Thus, acute administration of a nonintoxicating dose of MK-801 disrupts the retention of new information learned under the influence of the drug but does not interfere with the performance of tasks that are well learned before the drug is administered. Whether the performance deficits on the spatial learning tasks observed only following intoxicating doses of MK-801 reflect an effect on memory is not clear.     

Benzene-derived N-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA

Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N²-(4-Hydroxyphenyl)-2′-deoxyguanosine (N²-4-HOPh-dG) is the principal adduct identified in vivo by ³²P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N²-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N²-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson–Crick hydrogen bonds are present within the N²-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N²-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.     

Activation of distinct P2Y receptor subtypes stimulates insulin secretion in MIN6 mouse pancreatic β cells

Extracellular nucleotides and their receptor antagonists have therapeutic potential in disorders such as inflammation, brain disorders, and cardiovascular diseases. Pancreatic β cells express several purinergic receptors, and reported nucleotide effects on insulin secretion are contradictory. We studied the effect of P2Y receptors on insulin secretion and cell death in MIN6, mouse pancreatic β cells. Expression of P2Y₁ and P2Y₆ receptors was revealed by total mRNA analysis using RT-PCR. MIN6 cells were stimulated in the presence of 16.7 mM glucose with or without P2Y₁ and P2Y₆ agonists, 2-MeSADP and Up₃U, respectively. Both the agonists increased insulin secretion with EC₅₀ values of 44.6 ± 7.0 nM and 30.7 ± 12.7 nM respectively. The insulin secretion by P2Y₁ and P2Y₆ agonists was blocked by their selective antagonists MRS2179 and MRS2578, respectively. Binding of the selective P2Y₁ receptor antagonist radioligand [¹²⁵I]MRS2500 in MIN6 cell membranes was saturable (K_D 4.74 ± 0.47 nM), and known P2Y₁ ligands competed with high affinities. Inflammation and glucose toxicity lead to pancreatic β cell death in diabetes. Flow cytometric analysis revealed that Up₃U but not 2-MeSADP protected MIN6 cells against TNF-α induced apoptosis. Overall, the results demonstrate that selective stimulation of P2Y₁ and P2Y₆ receptors increases insulin secretion that accompanies intracellular calcium release, suggesting potential application of P2Y receptor ligands in the treatment of diabetes.     

Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A3 adenosine receptors

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A₃ receptors activation. We revealed that A₃ receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A₃ receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A₃ receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A₃ receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells.     

N6-Substituted adenosine derivatives: selectivity,efficacy, and species differences at A3 adenosine receptors

The activation of the human A(3) adenosine receptor (AR) by a wide range of N(6)-substituted adenosine derivatives was studied in intact CHO cells stably expressing this receptor. Selectivity of binding at rat and human ARs was also determined. Among N(6)-alkyl substitutions, small N(6)-alkyl groups were associated with selectivity for human A(3)ARs vs. rat A(3)ARs, and multiple points of branching were associated with decreased hA(3)AR efficacy. N(6)-Cycloalkyl-substituted adenosines were full (/=6 carbons) hA(3)AR agonists. N(6)-(endo-Norbornyl)adenosine 13 was the most selective for both rat and human A(1)ARs. Numerous N(6)-arylmethyl analogues, including substituted benzyl, tended to be more potent in binding to A(1) and A(3) vs. A(2A)ARs (with variable degrees of partial to full A(3)AR agonisms). A chloro substituent decreased the efficacy depending on its position on the benzyl ring. The A(3)AR affinity and efficacy of N(6)-arylethyl adenosines depended highly on stereochemistry, steric bulk, and ring constraints. Stereoselectivity of binding was demonstrated for N(6)-(R-1-phenylethyl)adenosine vs. N(6)-(S-1-phenylethyl)adenosine, as well as for the N(6)-(1-phenyl-2-pentyl)adenosine, at the rat, but not human A(3)AR. Interestingly, DPMA, a potent agonist for the A(2A)AR (K(i)=4nM), was demonstrated to be a moderately potent antagonist for the human A(3)AR (K(i)=106nM). N(6)-[(1S,2R)-2-Phenyl-1-cyclopropyl]adenosine 48 was 1100-fold more potent in binding to human (K(i)=0.63nM) than rat A(3)ARs. Dual acting A(1)/A(3) agonists (N(6)-3-chlorobenzyl- 29, N(6)-(S-1-phenylethyl)- 39, and 2-chloro-N(6)-(R-phenylisopropyl)adenosine 53) might be useful for cardioprotection.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.     

The in vitro effects of Trolox on methylmercury-induced neurotoxicity

Methylmercury (MeHg), an environmental toxicant primarily found in fish and seafood poses a dilemma to both consumers and regulatory authorities given the nutritional benefits of fish consumption vs. possible adverse neurological damage caused by MeHg. The present study addresses whether supplementation with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), alters the neuro-oxidative effects of MeHg in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) were measured. The cellular mercury (Hg) content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). The amount of MeHg-induced ROS was significantly reduced (p < 0.05) after treatment with 50 μM Trolox in C6 glial cell line. However, treatment with Trolox did not induce any significant increase in GSH levels or MTT activity in either of the cell lines. In addition, treatment with Trolox did not induce any significant changes in intracellular MeHg levels. The MeHg and Trolox treated C6 glial cell line differed significantly (p < 0.05) from the B35 cell line for MTT, ROS and GSH activity. These findings provide experimental evidence that preincubation with Trolox prevents MeHg-induced ROS generation in C6 glial cell line by quenching of free radicals and not by changes in intracellular GSH or MeHg content.     

Positive allosteric modulators at GABAB receptors exert intrinsic actions and enhance the influence of baclofen on light-induced phase shifts of hamster circadian activity rhythms

Light-induced phase shifts of hamster circadian activity rhythms are modulated by GABA_B receptors. Recently, positive allosteric modulators (PAM)s at GABA_B receptors were described, but it is not known whether they affect light-induced entrainment of circadian rhythms. Therefore, we studied the effects of two GABA_B PAMs, GS39783 and RacBHFF, upon light-induced phase advances and delays of hamster circadian wheel-running activity rhythms. Wheel running activity was recorded for Syrian hamsters maintained in constant darkness. Drugs administered intraperitoneally were evaluated for their ability to modulate a light-induced shift of the circadian activity rhythm. Baclofen (3.75-15 mg/kg) dose-dependently inhibited both light-induced phase advances and delays of hamster wheel running rhythms, and its actions were blocked by the selective GABA_B antagonist, SCH50911 (5 mg/kg). Neither GS39783 (3-30 mg/kg) nor RacBHFF (0.63-10 mg/kg) affected phase advances when injected alone, but both GS39783 (3 mg/kg) and RacBHFF (10 mg/kg) augmented the inhibitory effect of baclofen (5 mg/kg). At doses above 3 mg/kg, GS39783 and RacBHFF significantly inhibited phase delays alone, consistent with the notion of “agonist-allosteric” properties. GS39783 (0.5 mg/kg), but not RacBHFF (10 mg/kg), augmented the inhibitory action of baclofen on phase delays. These data are consistent with the possibility that GS39783 and RacBHFF act as PAMs at GABA_B receptors inhibiting light-induced phase advances, yet that they also posses “allosteric agonist” actions at the (presumably separate) population of GABA_B receptors modulating light-induced phase delays. GABA_B receptors clearly warrant further investigation as agents for modulation of circadian dysfunction associated with CNS disorders such as depression.     

Differential allosteric modulation by amiloride analogues of agonist and antagonist binding at A(1) and A(3) adenosine receptors

The diuretic drug amiloride and its analogues were found previously to be allosteric modulators of antagonist binding to A(2A) adenosine receptors. In this study, the possibility of the allosteric modulation by amiloride analogues of antagonist binding at A(1) and A(3) receptors, as well as agonist binding at A(1), A(2A), and A(3) receptors, was explored. Amiloride analogues increased the dissociation rates of two antagonist radioligands, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one ([3H]PSB-11), from A(1) and A(3) receptors, respectively. Amiloride and 5-(N,N-dimethyl)amiloride (DMA) were more potent at A(1) receptors than at A(3) receptors, while 5-(N,N-hexamethylene)amiloride (HMA) was more potent at A(3) receptors. Thus, amiloride analogues are allosteric inhibitors of antagonist binding at A(1), A(2A), and A(3) adenosine receptor subtypes. In contrast to their effects on antagonist-occupied receptors, amiloride analogues did not affect the dissociation rates of the A(1) agonist [3H]N(6)-[(R)-phenylisopropyl]adenosine ([3H]R-PIA) from A(1) receptors or the A(2A) agonist [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) from A(2A) receptors. The dissociation rate of the A(3) agonist radioligand [125I]N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]I-AB-MECA) from A(3) receptors was decreased significantly by amiloride analogues. The binding modes of amiloride analogues at agonist-occupied and antagonist-occupied receptors differed markedly, which was demonstrated in all three subtypes of adenosine receptors tested in this study. The effects of the amiloride analogues on the action of the A(3) receptor agonist were explored further using a cyclic AMP functional assay in intact CHO cells expressing the human A(3) receptor. Both binding and functional assays support the allosteric interactions of amiloride analogues with A(3) receptors.     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

A role for AMPA receptors in mood disorders

Major antidepressant agents increase synaptic levels of monoamines. Although the monoamine hypothesis of depression remains a cornerstone of our understanding of the pathophysiology of depression, emerging data has suggested that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype of glutamate receptor may also play a pivotal role in depression. Positive allosteric modulators of AMPA receptors increase brain levels of brain-derived neurotrophic factor (BDNF) that impacts the viability and generation of neurons in key brain structures. AMPA receptor potentiators are active in rodent models predictive of antidepressant efficacy. The mechanisms by which AMPA receptor potentiators produce these biological effects, however, are uncertain. Current evidence points to an antidepressant mechanism that is independent of monoaminergic facilitation that is driven by neurogenesis, a process facilitated by increased BDNF expression. However, alternative hypotheses need to be considered given uncertainties in the relationship between BDNF increases and the effects of conventional antidepressant medications. Electrophysiological and protein conformational data indicate that structural variants of AMPA receptor potentiators can differentially modulate AMPA receptor-mediated currents, although the manner in which this impacts antidepressant efficacy is yet to be understood. Conventional antidepressants such as fluoxetine positively modulate AMPA receptors. This potentiation is engendered by specific phosphorylation pathways activated through the dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32). Other novel compounds with antidepressant-like effects in rodents may also produce their in vivo effects through potentiation of AMPA receptors. Thus, AMPA receptor potentiation might be a general mechanism through which the clinical outcome of antidepressant efficacy is achieved.