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PubMed2006

Multi-process herbicide transport in structured soil columns: experiments and model analysis.

Model predictions of pesticide transport in structured soils are complicated by multiple processes acting concurrently. In this study, the hydraulic, physical, and chemical nonequilibrium (HNE, PNE, and CNE, respectively) processes governing herbicide transport under variably saturated flow conditions were studied. Bromide (Br-), isoproturon (IPU, 3-(4-isoprpylphenyl)-1,1-dimethylurea) and terbuthylazine (TER, N2-tert-butyl-6-chloro-N4-ethyl-1,3,5-triazine-2,4-diamine) were applied to two soil columns. An aggregated Ap soil column and a macroporous, aggregated Ah soil column were irrigated at a rate of 1 cm h(-1) for 3 h. Two more irrigations at the same rate and duration followed in weekly intervals. Nonlinear (Freundlich) equilibrium and two-site kinetic sorption parameters were determined for IPU and TER using batch experiments. The observed water flow and Br- transport were inversely simulated using mobile-immobile (MIM), dual-permeability (DPM), and combined triple-porosity (DP-MIM) numerical models implemented in HYDRUS-1D, with improving correspondence between empirical data and model results. Using the estimated HNE and PNE parameters together with batch-test derived equilibrium sorption parameters, the preferential breakthrough of the weakly adsorbed IPU in the Ah soil could be reasonably well predicted with the DPM approach, whereas leaching of the strongly adsorbed TER was predicted less well. The transport of IPU and TER through the aggregated Ap soil could be described consistently only when HNE, PNE, and CNE were simultaneously accounted for using the DPM. Inverse parameter estimation suggested that two-site kinetic sorption in inter-aggregate flow paths was reduced as compared to within aggregates, and that large values for the first-order degradation rate were an artifact caused by irreversible sorption. Overall, our results should be helpful to enhance the understanding and modeling of multi-process pesticide transport through structured soils during variably saturated water flow.

Bromides↗
PubMed2004

RDX biodegradation column study: comparison of electron donors for biologically induced reductive transformation in groundwater.

A series of column studies, using site-specific soil and groundwater, were conducted to determine the feasibility of biologically active zone enhancement (BAZE) process for reductive biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater. This treatability study examined the use of four amendments (acetate, ethanol, soluble starch, and acetate plus ammonium), which served as electron donors. Triplicate columns, with groundwater residence time of about 27.5 h, were used for each amendment treatment and the amendment control. In treatment columns amendment dosing was 500 mg/L C for carbon sources and 100 mg/L N for ammonium. Each of the amendment treatments reduced RDX inlet concentrations of 100 microg/L to less than 1 microg/L. The highest first-order RDX biodegradation rate ranged between 0.140 and 0.447 h(-1) for acetate amended columns as compared to 0.037 to 0.083 h(-1) in control columns (no amendment). The addition of soluble starch resulted in increased toxicity (based on Microtox analysis) that was partially removed by biological activity in the columns. Ethanol addition itself did not result in increased toxicity but biological activity in this system did induce Microtox toxicity. Acetate did not have any Microtox toxicity associated with it. The addition of ammonium as a nitrogen source did not significantly increase the removal rate of RDX. Based on these observations acetate was selected for the field demonstration.

Acetates↗
PubMed2005

Kinetics of RDX degradation by zero-valent iron (ZVI).

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common groundwater contaminant at military facilities. The current research has been conducted to evaluate the use of zero-valent iron (ZVI) for the remediation of water contaminated with RDX. RDX was found to degrade rapidly in the presence of ZVI. The observed first-order kinetic constant for RDX reduction follows an enzymatic-like kinetic model with respect to the ZVI concentration. At low ZVI concentrations, RDX reduction follows pseudo first order kinetics with respect to ZVI concentration; while at high ZVI concentrations the RDX reduction is zero-order. Nitroso compounds (MNX, DNX, and TNX), nitrate, nitrite and nitrous oxide were identified as the main by-products for the RDX reduction by ZVI. The nitroso compounds were found to undergo reduction by ZVI.

Iron↗
PubMed2004

Synthesis, crystal structure and DNA cleavage activities of copper(II) complexes with asymmetric tridentate ligands.

Two asymmetric tridentate copper(II) complexes, [Cu(dppt)Cl(2)].0.25H(2)O (1) (dppt=3-(1,10-phenanthrolin-2-yl)-5,6-diphenyl-as-triazine) and [Cu(pta)Cl(2)] (2) (pta=3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]acenaphthylene), have been prepared and characterized by elemental analysis, IR and Fast atomic bombardment mass spectra. Complex 1 has also been structurally characterized. The complexes exist as distorted square pyramid with five co-ordination sites occupied by the tridentate ligand and the two chlorine anions. DNA interaction studies suggest that the ligand planarity of the complex has a significant effect on DNA binding affinity increasing in the order [Cu(dppt)Cl(2)]< [Cu(pta)Cl(2)]. In the presence of ascorbate or glutathione, the two complexes are found to cause significant cleavage of double-strand pBR 322 DNA and [Cu(pta)Cl(2)] exhibited the higher cleaving efficiency.

Animals↗
PubMed2004

DNA interactions of cobalt(III) mixed-polypyridyl complexes containing asymmetric ligands.

Three novel asymmetric ligands, 3-(pyridine-2-yl)-5,6-diphenyl-as-triazine (pdtb), 3-(pyridine-2-yl)-as-triazino[5,6-f]acenaphthylene (pdta) and 3-(pyridine-2-yl)-as-triazino[5,6-f]phenanthroline (pdtp) and their cobalt(III) complexes have been synthesized and characterized. Binding of the three complexes with calf thymus DNA (CT-DNA) has been investigated by spectroscopic methods, viscosity, cyclic voltammetry, and electrophoresis measurements. The experimental results indicate that the size and shape of the intercalated ligand have a marked effect on the binding affinity of complexes to CT-DNA. Complexes 2 and 3 have also been found to promote cleavage of plasmid pBR322 DNA from the supercoiled form I to the open circular form II upon irradiation.

Acenaphthenes↗
PubMed2005

Cu(II) complexes with a sulfonamide derived from benzoguanamine. Oxidative cleavage of DNA in the presence of H2O2 and ascorbate.

Reaction between benzoguanamine (2,4-diamino-6-phenyl-1,3,5-triazine) and 2-mesitylenesulfonyl chloride leads to formation of a sulfonamide able to form two mononuclear Cu(II) complexes with a CuL(2) stoichiometry. The local environment of the metal cation is a distorted octahedron, with two ligands and two solvent molecules; both complexes crystallize in the monoclinic structure, space group P2(1), with Z=2. In the presence of ascorbate/H(2)O(2,) the two complexes significantly cleavage double-strand pUC18 DNA plasmid. Both complexes exhibit more nuclease efficiency that the copper phenantroline complex. From scavenging reactive oxygen studies we conclude that the hydroxyl radical and a singlet oxygen-like entity, such a peroxide copper complex, are the radical species involved in the DNA damage.

Ascorbic Acid↗
PubMed2004

Phosphodiesterase inhibition by a gastroprotective agent irsogladine: preferential blockade of cAMP hydrolysis.

The effect of irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate], an antiulcer drug, on contents of cyclic nucleotides including cAMP and cGMP was investigated in rat stomachs. Irsogladine concentration-dependently increased cAMP content in rat glandula stomach. However, irsogladine at higher concentration (10(-5) M) was unable to further increase cAMP level in the presence of non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine, although 3-isobutyl-1-methylxanthine by itself increased cAMP level. On the other hand, irsogladine had no effect on the glandula cGMP content. Subsequently, the effect of irsogladine on the cyclic nucleotide degradation by purified bovine brain and heart PDEs was investigated. The cAMP degradation by purified bovine brain PDE was partially suppressed by PDE1 inhibitor vinpocetin, PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride and PDE4 inhibitor rolipram but not by PDE3 inhibitor cilostamide, and completely inhibited by 3-isobutyl-1-methylxanthine, suggesting that is attributed almost exclusively to PDE1, PDE2 and PDE4. Meanwhile, cGMP degradation by purified bovine brain PDE was partially suppressed by erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Irsogladine preferentially inhibited the response to cAMP degradation compared with cGMP degradation by this brain PDE. The cAMP degradation by bovine heart PDE was almost completely inhibited by the combination with vinpocetine and cilostamide, indicating that is mediated almost exclusively by PDE1 and PDE3. Irsogladine suppressed this cAMP degradation measured in the presence of vinpocetine to almost the same extent as that determined in the presence of cilostamide. These results indicate that irsogladine produces the increase of intracellular cAMP content via non-selective inhibition of PDE isozymes, which may be a key mechanism involved in its gastroprotective actions.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Irsogladine, an anti-ulcer drug, suppresses superoxide production by inhibiting phosphodiesterase type 4 in human neutrophils.

Neutrophil superoxide production is implicated in the pathogenesis of gastric mucosal damage induced by various ulcerative agents and Helicobacter pylori infection. We investigated here the effects of an anti-ulcer drug irsogladine [2, 4-diamino-6-(2, 5-dichlorophenyl)-s-triazine maleate] on cAMP formation in isolated human neutrophils. The cAMP level in human neutrophils was elevated by a phosphodiesterase (PDE) type 4 selective inhibitor rolipram, but not by any inhibitors of PDE1, PDE2 and PDE3. Irsogladine also increased cAMP formation in a concentration-dependent manner in neutrophils. A non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) alone significantly increased cAMP level, whereas irsogladine was unable to further increase cAMP level in the presence of IBMX. Irsogladine inhibited concentration-dependently the superoxide (O(2)(-)) production induced by various stimuli including formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, guanosine 5'-[gamma-thio] triphosphate, A23187 and phorbol 12-myristate 13-acetate. These effects of irsogladine were mimicked by rolipram, IBMX and dibutyryl cAMP. The inhibitory effects of irsogladine and rolipram on the O(2)(-) production were reversed by a protein kinase A inhibitor H-89. These results indicate that irsogladine inhibits the superoxide production in human neutrophils by the increase of cAMP content by PDE 4 inhibition, which in turn contributing to the anti-ulcer effects of irsogladine on gastric mucosal lesions associated with oxidative stress.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Kinetic and functional properties of [3H]ZM241385, a high affinity antagonist for adenosine A2A receptors.

We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.

Adenosine↗
PubMedDate not supplied

Flow cytometry and pigment analyses as tools to investigate the toxicity of herbicides to natural phytoplankton communities.

Characterisation of natural phytoplanktonic communities is currently being advanced through flow cytometry and high resolution pigment analyses. To date, toxicological methods to assess impacts of herbicides on natural phytoplankton populations are lacking. Here, we report the novel use of these techniques in combination to study changes in phytoplankton populations exposed to 2-methylthio-4-tertiary-butylamino-6-cyclopropylamino-s-triazine (Irgarol 1051), a herbicide used in antifouling paints. Flow cytometry results revealed that following a 72-h exposure to approximately 100 ngL(-1), eukaryote abundance was less than half that in the controls. High performance liquid chromatographic analyses of pigments demonstrated that 19'-hexanoyloxyfucoxanthin was selectively lost relative to the control. This carotenoid is specific to the prymnesiophytes which are key constituents of phytoplanktonic communities within temperate marine waters. Values of EC(50) (72 h) as low as 70 ngL(-1) were calculated from the selective reduction in this compound. Concentrations substantially exceeding this level have been reported in UK and other European coastal waters.

Chromatography, High Pressure Liquid↗
PubMed2005

Contamination of Caribbean coastal waters by the antifouling herbicide Irgarol 1051.

Irgarol 1051 is a s-triazine herbicide used in popular slime-resistant antifouling paints. It has been shown to be acutely toxic to corals, mangroves and sea grasses, inhibiting photosynthesis at low concentrations (>50 ng l(-1)). We present the first data describing the occurrence of Irgarol 1051 in coastal waters of the Northeastern Caribbean (Puerto Rico (PR) and the US Virgin Islands (USVI)). Low level contamination of coastal waters by Irgarol 1051 is reported, the herbicide being present in 85% of the 31 sites sampled. It was not detected in water from two oceanic reference sites. In general, Irgarol 1051was present at concentrations below 100 ng l(-1), although far higher concentrations were reported at three locations within Benner Bay, USVI (223-1,300 ng l(-1)). The known toxicity of Irgarol 1051 to corals and sea grasses and our findings of significant contamination of the Northeastern Caribbean marine environment by this herbicide underscore the importance of understanding, more fully, local and regional exposure of reef and sea grass habitats to Irgarol 1051 and, where necessary, implementing actions to ensure adequate protection of these important ecosystems.

Animals↗
PubMed2006

A 3D hydrodynamic fate and transport model for herbicides in Sacca di Goro coastal lagoon (Northern Adriatic).

Sacca di Goro is a shallow coastal microtidal lagoon with a surface area of 26 km2, and an average depth of about 1.5m. Fresh water pollutant loads from Po River branches and several drainage canals lead to anthropogenic eutrophication, frequent summer anoxia crises and chemical contamination. Such events not only affect the lagoon ecosystem but also cause serious economic losses, the lagoon being the second largest producer of clams in Italy. The present work aims at using a fate model coupled with COHERENS 3D hydrodynamic model to simulate and to explain the spatial distribution and temporal variations of s-triazines herbicides in the Sacca di Goro lagoon. The simulation results of spatial and temporal dynamic behaviour of atrazine, simazine and terbuthylazine have been compared with experimental data obtained during an annual monitoring programme.

Animals↗
PubMed2004

Modulation of short-term social memory in rats by adenosine A1 and A(2A) receptors.

The recognition of an unfamiliar juvenile rat by an adult rat has been shown to imply short-term memory processes. The present study was designed to examine the role of adenosine receptors in the short-term social memory of rats using the social recognition paradigm. Adenosine (5.0-10.0 mg/kg), the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 0.025-0.05 mg/kg) and the selective adenosine A(2A) receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA, 1.0-5.0 mg/kg), given by i.p. route 30 min before the test, disrupted the juvenile recognition ability of adult rats. This negative effect of adenosine (5.0 mg/kg, i.p.) on social memory was prevented by pretreatment with the non-selective adenosine receptor antagonist caffeine (10.0 mg/kg, i.p.), the adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1.0 mg/kg, i.p.) and the adenosine A(2A) antagonist 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, 1.0 mg/kg, i.p.). Furthermore, acute administration of caffeine (10.0-30.0 mg/kg, i.p.), DPCPX (1.0-3.0 mg/kg, i.p.) or ZM241385 (0.5-1.0 mg/kg, i.p.) improved the short-term social memory in a specific manner. These results indicate that adenosine modulates the short-term social memory in rats by acting on both A1 and A(2A) receptors, with adenosine receptor agonists and antagonists, respectively, disrupting and enhancing the social memory.

Adenosine↗
PubMed2006

Mouse spinal cord compression injury is reduced by either activation of the adenosine A2A receptor on bone marrow-derived cells or deletion of the A2A receptor on non-bone marrow-derived cells.

Activation of the adenosine A(2A) receptor (A(2A)R) at the time of reperfusion has been shown to reduce ischemia-reperfusion injury in peripheral tissues and spinal cord. In this study we show that treating mice with the A(2A)R agonist, 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester for four days beginning before or just after the onset of reperfusion after compression-induced spinal cord injury rapidly (within 1 day) and persistently (>42 days) reduces locomotor dysfunction and spinal cord demyelination. Protection is abolished in knockout/wild type bone marrow chimera mice selectively lacking the A(2A)R only on bone marrow-derived cells but retaining receptors on other tissues including blood vessels. Paradoxically, reduced spinal cord injury is also noted in A(2A)R -/- mice, and in wild type/knockout bone marrow chimera mice selectively lacking the A(2A)R on non-bone marrow-derived cells, or in mice treated with the A(2A) antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol. The greatest protection is seen in knockout/wild type bone marrow chimera mice treated with 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester, i.e. by activating the A(2A)R in mice expressing the receptor only in bone marrow-derived cells. The data suggest that inflammatory bone marrow-derived cells are the primary targets of A(2A) agonist-mediated protection. We conclude that A(2A) agonists or other interventions that inhibit inflammation during and after spinal cord ischemia may be effective in reducing spinal cord injury in patients, but excessive or prolonged stimulation of the A(2A)R may be counterproductive. It may be possible to devise strategies to produce optimal spinal cord protection by exploiting temporal differences in A(2A)R-mediated responses.

Adenosine A2 Receptor Agonists↗
PubMed2004

Mixture additives inhibit the dermal permeation of the fatty acid, ricinoleic acid.

Ricinoleic acid (RA) like many of the ingredients in machine cutting fluids and other industrial formulations are potential dermal irritants, yet very little is known about its permeability in skin. 3H-ricinoleic acid mixtures were formulated with three commonly used cutting fluid additives; namely, triazine (TRI), linear alkylbenzene sulfonate (LAS), and triethanolamine (TEA) and topically applied to inert silastic membranes and porcine skin in vitro as aqueous mineral oil (MO) or polyethylene glycol (PEG) mixtures. These additives significantly decreased ricinoleic acid partitioning from the formulation into the stratum corneum (SC) in PEG-based mixtures. Except for LAS, all other additives produced a more basic formulation (pH = 9.3-10.3). In silastic membranes and porcine skin, individual additives or combination of additives significantly reduced ricinoleic permeability. This trend in ricinoleic acid disposition in both membranes suggests that the mixture interaction is more physicochemical in nature and probably not related to the chemical-induced changes in the biological membrane as may be assumed with topical exposures to potentially irritant formulations.

Alkanesulfonic Acids↗
PubMed2005

Zero-valent iron pretreatment for enhancing the biodegradability of RDX.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (C3H6N3(NO2)3, royal demolition explosive or RDX) is a common nitramine explosive and one of the major constituents in wastewaters from ammunitions plants. The objective of this study is to investigate zero-valent iron (Fe0) pretreatment for enhancing the biodegradability of recalcitrant RDX. It was hypothesized that iron pretreatment can reductively transform RDX to products that are more amenable to biological treatment processes such as activated sludge. Results of batch and column experiments showed rapid and complete removal of RDX by Fe0 regardless of the buffering capacity. Formaldehyde (HCHO), a major reduction product of RDX, was readily biodegraded by a mixed culture. Respirometric data indicate that iron-treated RDX solution exerted substantially higher biochemical oxygen demand (BOD) than untreated RDX solution. We propose that an integrated iron reduction-activated sludge process may be a feasible option for treating RDX-laden wastewater.

Biodegradation, Environmental↗
PubMed2002

Diaphorase catalyzed biotransformation of RDX via N-denitration mechanism.

Previously, we hypothesized that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be biotransformed by anaerobic sludge via three different routes: (1) direct ring cleavage via alpha-hydroxylation of a-CH(2) group, (2) reduction of one of the -NO(2) groups to -NO, (3) N-denitration prior to ring cleavage. The present study describes biotransformation of RDX via route 3 by a diaphorase (EC 1.8.1.4) from Clostridium kluyveri using NADH as electron donor. The removal of RDX was accompanied by the formation and accumulation of nitrite ion (NO(2)(-)), formaldehyde (HCHO), ammonium (NH(4)(+)), and nitrous oxide (N(2)O). None of the RDX-nitroso products were detected. The ring cleavage product methylenedinitramine was detected as the transient intermediate. Product stoichiometry showed that each reacted RDX molecule produced one nitrite ion and the product distribution gave a carbon (C) and nitrogen (N) mass balance of 91 and 92%, respectively, supporting the occurrence of a mono-denitration step prior to the ring cleavage and decomposition. Severe oxygen mediated inhibition (92% inhibition) of RDX biotransformation and superoxide dismutase-sensitive cytochrome c reduction indicated the potential involvement of an anion radical RDX(.-) prior to denitration. A comparative study between native- and apo-enzymes showed the possible involvement of flavin mononucleotide (FMN) in catalyzing the transfer of a redox equivalent (e/H(+)) from NADH to RDX to produce RDX(.-) responsible for secondary decomposition.

Catalysis↗
PubMed2000

Site-directed mutagenesis studies of human A(2A) adenosine receptors: involvement of glu(13) and his(278) in ligand binding and sodium modulation.

To provide insights into interactions between ligands and A(2A) adenosine receptors, site-directed mutagenesis was used to test the roles of a glutamic acid residue in the first transmembrane domain (Glu13) and a histidine residue in the seventh transmembrane domain (His278). The two residues, which have been suggested to be closely linked in molecular modeling studies, were mutated to glutamine (E13Q) and tyrosine (H278Y), respectively. Saturation experiments revealed that [(3)H]ZM241385 (4-2-[7-amino-2-(2-furyl)-1,2, 4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol) bound wild-type and mutant receptors in membranes from COS-7 cells expressing human A(2A) adenosine receptors with high affinity and low non-specific binding. It was found from the competition experiments that the affinity of the A(2A) adenosine receptor agonists for the mutant receptors was 3- to 200-fold lower than for the wild-type receptor. Among antagonist competitors of binding at E13Q and H278Y mutant receptors, there was variation in the affinity depending on their different structures, although changes were relatively minor (<3-fold) except in the case of theophylline, whose affinity was decreased approximately 20 times on the H278Y mutant. The possible involvement of the two residues in sodium ion regulation was also tested. The agonist competition curves for [(3)H]ZM241385 were shifted to the right in both wild-type and mutant receptors in the presence of 1 M sodium ions, but the extent of shift (2- to 27-fold) in wild-type receptor was generally larger than for the mutant receptors. Sodium ions also decreased [(3)H]ZM241385 dissociation from both wild-type and mutant receptors, being more influential on the former than the latter. The results suggest that the two closely linked residues Glu13 and His278 in A(2A) adenosine receptor are most important for agonist recognition and are partly responsible for the allosteric regulation by sodium ions.

Animals↗