A Randomized, Controlled Comparison of NCX 470 (0.021%, 0.042%, and 0.065%) and Latanoprost 0.005% in Patients With Open-angle Glaucoma or Ocular Hypertension: The Dolomites Study.

PRCIS: NCX 470 0.042% and 0.065% were statistically superior in intraocular pressure (IOP) lowering to latanoprost 0.005%, and NCX 470 0.021% was noninferior. All NCX 470 concentrations were safe and well tolerated. PURPOSE: The purpose of this study was to compare varying concentrations of NCX 470 (a nitric oxide-donating bimatoprost) to latanoprost in a dose-response safety and efficacy trial. PATIENTS AND METHODS: Adult patients with bilateral open-angle glaucoma or ocular hypertension were randomized to NCX 470 0.021% (n=111), 0.042% (n=108), 0.065% (n=107), or latanoprost 0.005% (n=107) once daily in the evening. IOP was measured at 8:00 am, 10:00 am, and 4:00 pm at weeks 1, 2, and 4. The primary efficacy endpoint was the reduction from baseline in mean diurnal IOP at week 4. Secondary efficacy endpoints included reductions from baseline in mean diurnal IOP at weeks 1 and 2, and reductions from baseline in time-matched IOP at 8:00 am, 10:00 am, and 4:00 pm at weeks 1, 2, and 4. Adverse events were evaluated. RESULTS: All concentrations of NCX 470 resulted in significant reductions of mean diurnal IOP. The 0.042% and 0.065% concentrations were statistically superior to latanoprost 0.005%, and 0.021% was noninferior to latanoprost for change from baseline in mean diurnal IOP at week 4. The 0.065% concentration was also superior to latanoprost by up to 1.4 mm Hg for reduction from baseline at 8:00 am, 10:00 am, and 4:00 pm at week 4. NCX 470 was safe and well tolerated; conjunctival hyperemia was the most frequently reported adverse event. CONCLUSIONS: NCX 470 demonstrated dose-dependent reductions in IOP. The 0.042% and 0.065% concentrations demonstrated significantly greater reductions from baseline in mean diurnal IOP than latanoprost 0.005% at week 4, suggesting that higher concentrations may show even greater efficacy.

P2Y2 receptor activation decreases blood pressure and increases renal Na⁺ excretion.

ATP and UTP are endogenous agonists of P2Y(2/4) receptors. To define the in vivo effects of P2Y(2) receptor activation on blood pressure and urinary excretion, we compared the response to INS45973, a P2Y(2/4) receptor agonist and UTP analog, in wild-type (WT) and P2Y(2) receptor knockout (P2Y(2)-/-) mice. INS45973 was administered intravenously as a bolus injection or continuous infusion to determine effects on blood pressure and renal function, respectively. Within seconds, bolus application of INS45973 (0.1 to 3 mg/kg body wt) dose-dependently decreased blood pressure in WT (maximum response -35 ± 2 mmHg) and to a similar extent in endothelial nitric oxide synthase knockout mice. By contrast, blood pressure increased in P2Y(2)-/- (maximum response +18 ± 1 mmHg) but returned to basal levels within 60 s. Continuous infusion of INS45973 (25 to 750 µg·min(-1)·kg(-1) body wt) dose-dependently increased urinary excretion of Na(+) in WT (maximum response +46 ± 15%) but reduced Na(+) excretion in P2Y(2)-/- (maximum responses of -45 ± 15%) mice. In renal clearance experiments, INS45973 did not affect glomerular filtration rate but lowered blood pressure and increased fractional excretion of fluid, Na(+), and K(+) in WT relative to P2Y(2)-/- mice. The blood pressure responses to INS45973 are consistent with P2Y(2) receptor-mediated NO-independent vasodilation and implicate responses to endothelium-derived hyperpolarizing factor, and P2Y(2) receptor-independent vasoconstriction, probably via activation of P2Y(4) receptors on smooth muscle. Systemic activation of P2Y(2) receptors thus lowers blood pressure and inhibits renal Na(+) reabsorption, effects suggesting the potential utility of P2Y(2) agonism in the treatment of hypertension.

Lipophilic modifications to dinucleoside polyphosphates and nucleotides that confer antagonist properties at the platelet P2Y12 receptor.

Platelet P2Y12 receptors play a central role in the regulation of platelet function and inhibition of this receptor by treatment with drugs such as clopidogrel results in a reduction of atherothrombotic events. We discovered that modification of natural and synthetic dinucleoside polyphosphates and nucleotides with lipophilic substituents on the ribose and base conferred P2Y12 receptor antagonist properties to these molecules producing potent inhibitors of ADP-mediated platelet aggregation. We describe methods for the preparation of these functionalized dinucleoside polyphosphates and nucleotides and report their associated activities. By analysis of these results and by deconstruction of the necessary structural elements through selected syntheses, we prepared a series of highly functionalized nucleotides, resulting in the selection of an adenosine monophosphate derivative (62) for further clinical development.

Adenosine analogues as inhibitors of P2Y12 receptor mediated platelet aggregation.

Modified adenosine derivatives may lead to the development of P2Y(12) antagonists that are potent, selective, and bind reversibly to the receptor. Analogues of 2',3'-trans-styryl acetal-N6-ureido-adenosine monophosphate were prepared by modification of the 5'-position. The resulting analogues were tested for P2Y(12) antagonism in a platelet aggregation assay.

Synthesis of potential purinoceptor antagonists: application of P1-tBU phosphazene base for alkylation of adenine in solution and on solid phase.

Alkylation of adenine in solution and on solid phase was accelerated by phosphazene base P1-tBu compared to mineral bases. The reactions in solution afforded regioselectively the appropriate N9-alkylated adenines with high preparative yields while the reaction with polystyrene resin-bound N-bromoacetylated peptides gave three regioisomers (alkylated at the N9, N7, and N3 position of adenine) in a 4:2:1 molar ratio. Ten novel nonphosphate nucleotide analogues were tested in an ADP-induced platelet aggregation assay.

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family.

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.

Adenine nucleotide analogues locked in a Northern methanocarba conformation: enhanced stability and potency as P2Y(1) receptor agonists.

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5'-triphosphate agonists at P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors, but not P2Y(6) receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y(1) or human P2Y(1) and P2Y(2) receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC(50) at the hP2Y(1) receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5'-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N(6)-Me analogue were also highly selective, full agonists at P2Y(1) receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y(1) receptors, while the corresponding ribosides were inactive. Although beta,gamma-methylene-ATP was inactive at P2Y receptors, beta,gamma-methylene-(N)-methanocarba-ATP was a potent hP2Y(1) receptor agonist with an EC(50) of 160 nM and was selective versus hP2Y(2) and hP2Y(4) receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5'-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y(1) receptors.

Methanocarba modification of uracil and adenine nucleotides: high potency of Northern ring conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but not P2Y6 receptors.

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.

2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist.

1. We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y(1) receptor and that modification of the ribose in these analogues is tolerated in the P2Y(1) receptor binding pharmacophore. 2. Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety. 3. MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pK(B)=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y(1) receptor (pK(B)=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y(1) receptor since MRS2279 had no effect on activation of the human P2Y(2), P2Y(4), P2Y(6), or P2Y(11) receptors by their cognate agonists. 4. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. 5. In contrast, the P2Y(1) receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affinity (pK(B)=8.05) similar to that observed at the human P2Y(1) receptor heterologously expressed in 1321N1 cells. 6. Taken together these results illustrate selective high affinity antagonism of the P2Y(1) receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.

Avian and Human Homologues of the P2Y1 Receptor: Pharmacological, Signaling, and Molecular Properties.

[Table: see text] The P2Y receptor on turkey erythrocyte membranes was the first P2 receptor to be shown to activate phospholipase C (PLC) in a strictly guanine nucleotide-dependent manner and remains the only G protein-coupled P2 receptor for which G protein-coupling kinetics have been defined. This membrane receptor has provided a model system for detailed pharmacological analyses of a series of chain-extended 2-thioether derivatives of adenine nucleotides that exhibit remarkable selectivity and potency for P2Y receptors. This model system also has led recently to identification of a novel series of P2 receptor antagonists. The turkey erythrocyte receptor is the species homologue of the chick P2Y1 receptor originally cloned by Webb and coworkers [Webb et al., 1993]. We also have cloned the human homologue of the P2Y1 receptor, which exhibits identical pharmacological and second messenger signaling properties to that of the avian P2Y1 receptor.

Structure Activity Relationships for Derivatives of Adenosine-5'-Triphosphate as Agonists at P(2) Purinoceptors: Heterogeneity Within P(2X) and P(2Y) Subtypes.

The structure-activity relationships for a variety of adenine nucleotide analogues at P(2x)- and P(2Y)-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N(6)-substituents, and a uridine base instead of adenine), the ribose moiety (2' and 3'-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P(2Y)-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P(2X)-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P(2X)- or the P(2Y)-purinoceptors. Certain analogues displayed selectivity or specificity within the P(2X)- or P(2Y)-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2',3'-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P(2Y)-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P(2X)-purinoceptors. The potent agonist N(6)-methyl ATP and the somewhat less potent agonist 2'-deoxy-ATP were selective for P(2Y)-purinoceptors in the guinea pig taenia coli, but were inactive at P(2X)-purinoceptors and the vascular P(2Y)-purinoceptors. 3'-Benzylamino-3'-deoxyATP was very potent at the P(2X)-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P(2Y) receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P(2X)- and P(2Y)-purinoceptor classes.

Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal-5'-phosphate-6-phenylazo-2',4'-disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6-naphthylazo derivatives were also evaluated. Among the 6-phenylazo derivatives, 5'-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y(1) receptors, in guinea-pig vas deferens and bladder P2X(1) receptors, and in ion flux experiments by using recombinant rat P2X(2) receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X(1) receptors in differentiated HL-60 cell membranes was carried out by using [(35)S]ATP-γ-S. A 2'-chloro-5'-sulfo analog of PPADS (C(14)H(12)O(9)N(3)ClPSNa), a vinyl phosphonate derivative (C(15)H(12)O(11)N(3)PS(2)Na(3)), and a naphthylazo derivative (C(18)H(14)O(12)N(3)PS(2)Na(2)), were particularly potent in binding to human P2X(1) receptors. The potencies of phosphate derivatives at P2Y(1) receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C(15)H(13)O(8)N(3)PNa and its m-chloro analog C(15)H(12)O(8)N(3)ClPNa, which were selective for P2X vs. P2Y(1) receptors. C(15)H(12)O(8)N(3)ClPNa was very potent at rat P2X(2) receptors with an IC(50) value of 0.82 µM. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5-yl]methylphosphonic acid (C(14)H(12)-O(8)N(3)ClPSNa) showed high potency at P2Y(1) receptors with an IC(50) of 7.23 µM. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y(1) receptors, whereas at recombinant P2X(2) receptors had an IC(50) value of 1.1 µM. An ethyl phosphonate derivative (C(15)H(15)O(11)N(3)PS(2)Na(3)), whereas inactive at turkey erythrocyte P2Y(1) receptors, was particularly potent at recombinant P2X(2) receptors.

Transformation by a nucleotide-activated P2Y receptor is mediated by activation of Galphai, Galphaq and Rho-dependent signaling pathways.

BACKGROUND: Nucleotide-actived P2Y receptors play critical roles in the growth of tumor cells by regulating cellular proliferation, differentiation and survival. RESULTS: Here we demonstrate that an avian P2Y purinoceptor (tP2YR) with unique pharmacological and signal transduction properties induces morphologic and growth transformation of rodent fibroblasts. tP2YR induced a transformed phenotype similar to the mas oncogene, a G protein-coupled receptor which causes transformation by activation of Rac-dependent pathways. tP2YR-transformed cells exhibited increased steady-state activation of Rac1 and RhoA. Like activated Rho GTPases, tP2YR cooperated with activated Raf and caused synergistic transformation of NIH3T3 cells. Our data indicate that the ability of tP2YR to cause transformation is due to its unique ability among purinergic receptors to simultaneously activate Galphaq and Galphai. Co-expression of constitutively activated mutants of these two Galpha subunits caused the same transformed phenotype as tP2YR and Mas. Furthermore, transformation by both tP2YR and Mas was blocked by pharmacological inhibition of GalphaI by pertussis toxin (PTX) indicating an essential role for Galphai in transformation by these G-protein coupled receptors. CONCLUSIONS: Our data suggest that coordinated activation of Galphaq and Galphai may link the tP2YR and possibility the Mas oncogene with signaling pathways resulting in activation of Rho family proteins to promote cellular transformation.

Ocular surface distribution and pharmacokinetics of a novel ophthalmic 1% azithromycin formulation.

PURPOSE: To investigate the ocular distribution of 1% azithromycin ophthalmic solution and the effect of polycarbophil-based mucoadhesive formulation on ocular tissue levels of azithromycin after single and multiple topical administrations in the rabbit eye. METHODS: Rabbits were treated with either a single administration of 1% azithromycin solution with or without polycarbophil, or with multiple administrations of 1% azithromycin solution in polycarbophil. Drug concentrations were measured using LC/MS/MS. Conjunctiva, cornea, aqueous humor, and tear samples were analyzed over a period of 144 h after a single administration of azithromycin with or without polycarbophil. Eyelid, conjunctiva, cornea, aqueous humor, and tear samples were collected over a period of 288 h during and after multiple administrations of azithromycin. RESULTS: Azithromycin was rapidly absorbed and distributed in the ocular tissues, reaching within 5 min, concentrations of 10,539 microg/mL in tear film, 108 microg/g in conjunctiva, and 40 microg/g in the cornea. The drug demonstrated tissue-specific half-lives of 15, 63, and 67 h, respectively. Following multiple administrations, the drug gradually accumulated. The polycarbophil formulation increased the bioavailability of the drug, producing peak concentrations that were between 5- and 12-fold higher than those without polycarbophil. Azithromycin also distributed rapidly in the eyelids, reaching peak concentrations of 180 mug/g at the end of the 7-day treatment, and was eliminated with a half-life of 125 h. Six days after treatment was discontinued, eyelid levels of azithromycin were above 40 microg/g. CONCLUSIONS: Sustained and high concentrations were encountered with 7-day approved administration of 1% azithromycin formulation (AzaSite, Inspire Pharmaceuticals, Inc., Durham, NC) within all ocular surface tissues, particularly the lids. Many ocular surface disorders involving the tear film, eyelids, and adnexal structures are associated with chronic, low-grade bacterial infection and may potentially lead to decreased vision secondary to corneal scarring. Various topical antibiotic and steroid combinations with or without oral tetracyclines are commonly used with variable clinical response and known potential side effects. The clinical relevance of this study is unknown; however, the long-lasting antibacterial and additional anti-inflammatory properties of topical azithromycin might offer an effective alternative treatment option and should be explored further in clinical studies.

Rapid and reversible modulation of platelet function in man by a novel P2Y(12) ADP-receptor antagonist, INS50589.

P2Y(12) receptors participate in ADP-induced activation and aggregation of human platelets. INS50589, a selective P2Y(12) receptor antagonist, is being developed for use where controlled, reversible modulation of the platelet hemostatic function is needed. The tolerability, pharmacokinetics, and pharmacodynamics of INS50589 were tested in healthy human volunteers. Thirty-six subjects received intravenous infusions of placebo or INS50589 at 0.1-3 mg/kg/h for four hours. Platelet function, clotting parameters, bleeding time, safety assessments, and plasma concentrations of INS50589 and its major metabolite were monitored for 24 hours. Near-steady state plasma concentrations of INS50589 and effects on platelet function were achieved rapidly. The average maximal plasma concentration of INS50589 was linearly related to the dose administered. Intravenous INS50589 produced dose-dependent inhibition of platelet activation and aggregation in response to ADP in vitro until nearly full inhibition was achieved at the higher doses. Bleeding time was correspondingly increased, without any effect on activated clotting time, prothrombin time, or activated partial thromboplastin time. Platelet response to ADP had returned to at least 75% of the baseline value within 0.25-4 h after cessation of the intravenous infusion of INS50589, depending upon the dose and ADP challenge concentration. Infusions were well tolerated up to the highest dose tested. There was no evidence that the principal metabolite (INS51088) contributed to these effects. INS50589 is a well-tolerated, reversible, competitive antagonist of ADP at the P2Y(12) human platelet receptor, and its potential therapeutic utility in various cardiovascular settings is discussed.

International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy.

There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors de-orphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review.

Regulation of P2Y1 receptor-mediated signaling by the ectonucleoside triphosphate diphosphohydrolase isozymes NTPDase1 and NTPDase2.

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) control the concentration of released extracellular nucleotides, but the precise physiological roles played by these isozymes in modulation of P2 receptor signaling remain unclear. Activation of the human P2Y(1) receptor was studied in the presence of NTPDase1 or NTPDase2 expressed either in the same cell as the receptor or in P2Y(1) receptor-expressing cells cocultured with NTPDaseexpressing cells. Coexpression of NTPDase1 with the P2Y(1) receptor resulted in increases in the EC(50) for 2'-methylthioadenosine 5'-diphosphate (2MeSADP; 12-fold), ADP (50-fold), and ATP (10-fold) for activation of phospholipase C. Similar effects were observed when the P2Y(1) receptor and NTPDase1 were expressed on different cells. These results are explained by the capacity of NTPDase1 to hydrolyze both nucleoside triphosphates and diphosphates. NTPDase2 preferentially hydrolyzes nucleoside triphosphates, and the presence of NTPDase2 under either coexpression or coculture conditions did not change the EC(50) of 2MeSADP, ADP, or adenosine 5'-O-(2-thiodiphosphate) for activation of the P2Y(1) receptor. However, the EC(50) for ATP was 15-fold lower in the presence of NTPDase2 than in cells expressing the P2Y(1) receptor alone. Whereas expression of NTPDase1 decreased basal activity of the P2Y(1) receptor, the presence of the NTPDase2 resulted in P2Y(1) receptor-dependent increases in basal activity. These results suggest that basal activity of the P2Y(1) receptor is maintained by paracrine or autocrine release of receptor agonists and that the biological and/or pharmacological response mediated by P2Y receptors in target tissues is highly dependent on the types of ectonucleotidases expressed in the vicinity of the receptor.

Structure-activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors.

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y(1) while uridine containing dinucleotides have some level of agonist response on P2Y(2) and P2Y(6). The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.

Requirement of Cys399 for processing of the human ecto-ATPase (NTPDase2) and its implications for determination of the activities of splice variants of the enzyme.

Ecto-ATPase (CD39L1) corresponds to the type 2 enzyme of the ecto-nucleoside triphosphate diphosphohydrolase family (E-NTPDase). We have isolated from human ECV304 cells three cDNAs with high homology with members of the E-NTPDase family that encode predicted proteins of 495, 472, and 450 amino acids. Sequencing of a genomic DNA clone confirmed that these three sequences correspond to splice variants of the human ecto-ATPase (NTPDase2 alpha,-2 beta, and -2 gamma). Although all three enzyme forms were expressed heterologously to similar levels in Chinese hamster ovary cells clone K-1 (CHO-K1) cells, only the 495-amino acid protein (NTPDase2 alpha exhibited ecto-ATPase activity. Immunolocalization studies demonstrated that NTPDase2 alpha is fully processed and trafficked to the plasma membrane, whereas the NTPDase2 beta and -2 gamma splice variants were retained in not fully glycosylated forms in the endoplasmic reticulum. The potential roles of two highly conserved residues, Cys399 and Asn443, in the activity and cellular trafficking of the ecto-ATPase were examined. Mutation of Cys399, which is absent in NTPDase2 beta and -2 gamma, produced a protein completely devoid of nucleotidase activity, while mutation of Asn443 to Asp resulted in substantial loss of activity. Neither the Cys399 nor Asn443 mutants were fully glycosylated, and both were retained in the endoplasmic reticulum. These results indicate that the lack of ecto-nucleotidase activity exhibited by NTPDase2 beta and -2 gamma and the C399S mutant, as well as the large reduction of activity in the N443D mutant are due to alterations in the folding/maturation of these proteins.