A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.     

Enhanced expression of G protein-coupled receptor kinase 2 selectively increases the sensitivity of A2A adenosine receptors to agonist-induced desensitization

1. G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor (GPCR) responses. We have previously shown that stable expression of a dominant negative mutant G protein- coupled receptor kinase 2 (GRK2) construct in NG108-15 mouse neuroblastoma x rat glioma cells suppresses the agonist-induced desensitization of A_2A and A_2B adenosine receptor-stimulated adenylyl cyclase activity (Mundell et al., 1997). To further determine the role of GRK2 in agonist-induced desensitization of these adenosine receptors, we stably overexpressed wild type GRK2 in NG108-15 cells.
2. In homogenates prepared from cells overexpressing GRK2, the acute stimulation of adenylyl cyclase by activation of A_2A and A_2B adenosine receptors was markedly reduced, but could be reversed by pretreating the cells with AD (adenosine deaminase), to remove extracellular adenosine from the medium. On the other hand, acute stimulation of adenylyl cyclase by secretin, iloprost, NaF and forskolin was the same in GRK2 overexpressing cells and plasmid-transfected control cells.
3. Cells overexpressing GRK2 were more sensitive to adenosine receptor agonist-induced desensitization than plasmid-transfected control cells. This effect was selective since the agonist sensitivity of desensitization for secretin and IP-prostanoid receptor-stimulated adenylyl cyclase activity was not affected by GRK2 overexpression.
4. These results further implicate GRK2 as the likely mechanism by which A₂ adenosine receptors undergo short-term desensitization in NG108-15 cells, and indicate that even when overexpressed, GRK2 retains its substrate specificity for native receptors in intact cells. Furthermore, the susceptibility of GPCRs to desensitization appears to depend on the level of GRK expression, such that in cells that express high levels of GRK2, low agonist concentrations may be sufficient to trigger GRK-mediated desensitization.

     

Ser352 and Ser354 in the carboxyl terminus of the human P2Y(1) receptor are required for agonist-promoted phosphorylation and internalization in MDCK cells

BACKGROUND AND PURPOSE

The P2Y₁ receptor promotes chloride secretion in epithelial cells, a process critical for regulation of extracellular ion and fluid levels. Here we have examined the role of phosphorylation in agonist-induced internalization of P2Y₁ receptors.

EXPERIMENTAL APPROACH

A high-affinity radiolabelled antagonist, MRS2500, was used to quantify cell surface-binding sites of P2Y₁ receptors in Madin-Darby canine kidney (MDCK) epithelial cells, following exposure to agonists. The regions in the carboxyl terminus involved in both agonist-induced internalization of the receptor and its phosphorylation were identified by mutational analysis.

KEY RESULTS

Endogenous and stably expressed recombinant P2Y₁ receptors rapidly internalized with similar time courses in response to agonist in MDCK cells, ensuring that the levels of recombinant receptor achieved by retroviral infection did not adversely affect function of the internalization machinery. Four protein kinase C inhibitors of varying specificity did not affect internalization of recombinant receptors. Agonist-promoted internalization of a series of truncated P2Y₁ receptors identified a region between residues 349 and 359 in the carboxyl terminus as critical for regulation. Two amino acids within this region, Ser352 and Ser354, were shown to be both necessary and sufficient for agonist-promoted receptor phosphorylation and internalization.

CONCLUSIONS AND IMPLICATIONS

Our results firmly establish Ser352 and Ser354 in the carboxyl terminus of P2Y₁ receptors as critical residues for agonist-induced receptor internalization in MDCK cells. As the mechanism mediating this regulation requires phosphorylation of these key residues, the relevant receptor-regulated protein kinase can now be identified.     

Novel consequences of voltage-dependence to G-protein-coupled P2Y1 receptors

BACKGROUND AND PURPOSE: Emerging evidence suggests that activation of G-protein-coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non-excitable megakaryocyte using a range of agonists and antagonists. EXPERIMENTAL APPROACH: Simultaneous whole-cell patch clamp and fura-2 fluorescence recordings of rat megakaryocytes, which lack voltage-gated Ca2+ influx, were used to examine the voltage-dependence of P2Y1 receptor-evoked IP3-dependent Ca2+ mobilization. RESULTS: Depolarization transiently and repeatedly enhanced P2Y1 receptor-evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization-evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near-threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2'-pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization-evoked Ca2+ responses during co-application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP-induced voltage-dependent Ca2+ release. CONCLUSIONS AND IMPLICATIONS: The ability of depolarization to potentiate GPCRs at near-threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage-dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.     

Dependence of P2-nucleotide receptor agonist-mediated endothelium-independent relaxation on ectonucleotidase activity and A2A-receptors in rat portal vein

1. The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle.
2. At 25°C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-ClATP (2.7±0.5 μM, n=7)>ATP (12.9±1.1 μM, n=9)⩾2-MeSATP (21.9±4.8 μM, n=4). In the presence of αβ-methylene ATP (αβ-MeATP, 200 μM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited.
3. The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 100 μM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP.
4. The A_2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1–100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited.
5. ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to αβ-MeATP (200 μM) but were inhibited by ZM 241385 (100 nM).
6. Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [γ-³²P]-ATP showed that muscular strips rapidly (10–60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57±11% (n=3) by 200 μM αβ-MeATP.
7. These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A_2A-receptors.

     

Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate

The antagonist activity of N⁶-methyl 2′-deoxyadenosine 3′,5′-bisphosphate (N6MABP) has been examined at the phospholipase C-coupled P2Y₁ receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP-stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3′,5′-bisphosphate. The P2Y₁ receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pK_B=6.99±0.13). Whereas N6MABP was an antagonist at the human P2Y₁ receptor, no antagonist effect of N6MABP was observed at the human P2Y₂, human P2Y₄ or rat P2Y₆ receptors.     

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y₄ (hP2Y₄) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y₄ receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2-MeSATP=Ap₄A>UDP(pure). ADP, ATPγS, 2-MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y₄ is more like the P2U receptor subtype. The rP2Y₄ receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y₂ receptor (a known P2U receptor). Thus, rP2Y₄ and the P2Y₂ subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi-active) but can be distinguished as suramin-insensitive and suramin-sensitive P2U receptors, respectively.     

Tonic adenosine A1 and A2A receptor activation is required for the excitatory action of VIP on synaptic transmission in the CA1 area of the hippocampus

Adenosine can regulate synaptic transmission through modulation of the action of other neurotransmitters. The influence of adenosine on VIP enhancement of synaptic transmission in hippocampal slices was investigated. Facilitation of fEPSP slope by 1 nM VIP (23.3+/-1.3%) was turned into an inhibition (-12.1+/-3.4%) when extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1U/ml). Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM) or of A(2A) receptors with ZM241385 (20 nM) attenuated the effect of VIP. When both DPCPX and ZM241385 were present the effect of VIP was abolished. In the presence of ADA, selective A(1) receptor activation with N(6)-cyclopentyladenosine (CPA, 15 nM) or A(2A) receptor-activation with CGS21680 (10 nM) partially readmitted the excitatory effect of VIP on fEPSPs. In contrast, facilitation of PS amplitude by 1 nM VIP (19.1+/-1.2%) was attenuated in the presence of ADA or DPCPX but was not changed by ZM241385. CPA, in the presence of ADA, fully restored the effect of VIP on PS amplitude. In conclusion, VIP facilitation of synaptic transmission to hippocampal pyramidal cell dendrites is dependent on both A(1) and A(2A) receptor activation by endogenous adenosine. VIP effects on PS amplitude are only dependent on A(1) adenosine receptor activation. This differential sensitivity to adenosine modulation might be due to the different VIP circuits contributing to VIP effects on pyramidal cell dendrites and pyramidal cell bodies.     

Diastereoselectivity of the P2Y11 nucleotide receptor: mutational analysis

Background and purpose:

The P2Y₁₁ receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of P_α-substituted ATP derivatives (ATP-α-S isomers). These compounds are suitable candidates for the development of selective P2Y₁₁ receptor agonists that might be used as immune modulators. We have analysed the binding mode of ATP at the P2Y₁₁ receptor by molecular modeling and site-directed mutagenesis. Based on our recent findings, we decided to decipher the molecular determinants of stereoselective recognition at the P2Y₁₁ receptor.

Experimental approach:

Two amino acid residues [Glu186 in the extracellular loop 2 and Arg268 in the transmembrane domain 6 (TM6)], which are part of the nucleotide-binding pocket, were selected and studied by mutational analyses. We expected these residues to be involved in determining the stereospecificity of the P2Y₁₁ receptor.

Key results:

After mutation of Arg268 to alanine or glutamine, the stereospecific recognition of the ATP-α-S isomers at the P2Y₁₁ receptor was lost. In contrast, at the Glu186Ala receptor mutant, the stereoselective differentiation between these isomers was increased. On the Arg268Gln/Glu186Ala double mutant we observed no further effect, except for additivity in the decrease in potency of both isomers, as compared with the single-point mutants.

Conclusions and implications:

Our results show that the stereospecificity of the P2Y₁₁ receptor for P_α-substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands.     

ERK1/2 activation is involved in the neuroprotective action of P2Y13 and P2X7 receptors against glutamate excitotoxicity in cerebellar granule neurons

Cerebellar granule neurons express several types of nucleotide receptors, with the metabotropic P2Y₁₃ and the ionotropic P2X7 being the most relevant in this model. In the present study we investigated the role of P2Y₁₃ and P2X7 nucleotide receptors in ERK1/2 signalling. The nucleotidic agonists 2MeSADP (2-methylthioadenosine-5′-diphosphate) for P2Y₁₃ and BzATP (2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate) for P2X7 receptors were coupled to ERK1/2 activation in granule neurons, being able to increase around two-fold the levels of ERK1/2 phosphorylation. These effects were sensitive to the inhibitory action of the antagonists MRS-2211 and A-438079, specific for P2Y₁₃ and P2X7 receptors, respectively. Although both receptor subtypes shared the same pattern of transient ERK1/2 phosphorylation, they differed in the intracellular cascades they triggered, being PI3K-dependent for P2Y₁₃ and calcium/calmodulin kinase II (CaMKII)-dependent for P2X7. These two different ERK-mediated pathways were involved in the neuroprotective effects displayed by both P2Y₁₃ and P2X7 receptors against apoptosis induced by an excitotoxic concentration of glutamate, in a similar manner to the neurotrophin, BDNF. In addition, P2Y₁₃ and P2X7 receptor agonists were also able to phosphorylate and activate the ERK-dependent target CREB, which could be involved in their neuroprotective effect. These results indicate that nucleotide receptors share with trophic factors the same survival routes in neurons, such as the ERK signalling route, and therefore, can contribute to the maintenance of granule neurons in conditions in which survival is being compromised.     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM).
2. In the prostatic half of the vas deferens the A₁ selective adenosine receptor agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A₁/A₂ adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC₅₀±s.e.mean values 6.15±0.24, 5.99±0.26 and 5.51±0.24, respectively). The responses to CPA were blocked by the A₁ adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM).
3. In the epididymal half of the vas deferens NECA potentiated (at ⩽100 nM) and inhibited (at ⩾1 μM) electrically-evoked contractions. In the presence of the non-selective α-adrenoceptor antagonist phentolamine (3 μM), the α₁-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC₅₀ values 6.05±0.25, 5.97±0.29 and 5.71±0.27, respectively). CPA (at 10 μM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC₅₀ 6.14±0.67); this effect was antagonized by DPCPX (30 nM, apparent pK_B 8.26±0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 μM), CPA (up to 1 μM) potentiated electrically-evoked contractions.
4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC₅₀ values 7.49±0.62, 7.65±0.74 and 5.84±0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK_B value of 7.64±0.64.
5. The α₁-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 μM) potentiated responses to phenylephrine (⩽1 μM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 μM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 μM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC₅₀ values 7.57±0.54 and 8.08±0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis.
6. These studies are consistent with the action of stable adenosine analogues at prejunctional A₁ and postjunctional A₁-like adenosine receptors. The prejunctional A₁ adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of α₁-adrenoceptor-, but not of ATP-induced contractility.

     

Effects of P2Y(1) receptor antagonism on the reactivity of platelets from patients with stable coronary artery disease using aspirin and clopidogrel

BACKGROUND AND PURPOSE

P2Y₁ is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy.

EXPERIMENTAL APPROACH

Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day⁻¹) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y₁ inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed.

KEY RESULTS

Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and α_IIbβ₃ activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited α_IIbb₃ activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s⁻¹), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y₁₂ inhibitor 2-MeSAMP.

CONCLUSIONS AND IMPLICATIONS

Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y₁ in the blood of patients with CAD appears to play a more important role than previously anticipated.     

Rolofylline,an adenosine A1 receptor antagonist,inhibits osteoclast differentiation as an inverse agonist

BACKGROUND AND PURPOSE

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5′-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A₁ receptors (A₁R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A₁R agonists neither affect basal osteoclastogenesis nor do they reverse A₁R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A₁R antagonist-mediated inhibition, and, when we saw no effect, determined whether A₁R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.

EXPERIMENTAL APPROACH

Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand–macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.

KEY RESULTS

Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC₅₀ = 20–70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.

CONCLUSIONS AND IMPLICATIONS

Based on these findings, we hypothesize that the A₁R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A₁R antagonists act as inverse agonists to release A₁R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A₁R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.     

Quinolinedione nucleus as a novel scaffold for A1 and A2A adenosine receptor antagonists

In the last few years, much effort has been directed towards the synthesis of selective adenosine receptor (AR) antagonists since they are attractive tools for pharmacological intervention in many pathophysiological conditions. During our studies aimed at obtaining new nonclassical adenosine antagonists devoid of phosphodiesterase (PDE) inhibition, a series of 2-pyridones and 2,5-quinolinediones (3a–f, 5a–f, 6a,c–f) has been synthesized as potential AR ligands. Binding affinities of the new compounds were determined for bovine and human adenosine A₁, A_2A, and A₃ receptors. Compound 5f showed good affinity (K _i = 7.8 μM) towards human A₁AR but no selectivity (K _i = 7.0 μM) towards human A_2AAR, whereas compound 6f showed more affinity towards human A_2A (K _i = 16 μM) than A₁ receptor (percentage inhibition at 10 μM concentration = 11). In the 1–100 μM range, the new compounds did not inhibit cardiac PDE3 activity at all. Molecular modeling studies carried out on 5f and 6f support the pharmacological results and suggest 6f as a potential lead compound selective towards A_2AAR.     

Treating lung inflammation with agonists of the adenosine A2A receptor: promises, problems and potential solutions

Adenosine A(2A) receptor agonists may be important regulators of inflammation. Such conclusions have come from studies demonstrating that, (i) adenosine A(2A) agonists exhibit anti-inflammatory properties in vitro and in vivo, (ii) selective A(2A) antagonists enhance inflammation in vivo and, (iii) knock outs of this receptor aggravate inflammation in a wide variety of in vivo models. Inflammation is a hallmark of asthma and COPD and adenosine has long been suggested to be involved in disease pathology. Two recent publications, however, suggested that an inhaled adenosine A(2A) receptor agonist (GW328267X) did not affect either the early and late asthmatic response or symptoms associated with allergic rhinitis suggesting that the rationale for treating inflammation with an adenosine A(2A) receptor agonist may be incorrect. A barrier to fully investigating the role of adenosine A(2A) receptor agonists as anti-inflammatory agents in the lung is the side effect profile due to systemic exposure, even with inhalation. Unless strategies can be evolved to limit the systemic exposure of inhaled adenosine A(2A) receptor agonists, the promise of treating lung inflammation with such agents may never be fully explored. Using strategies similar to that devised to improve the therapeutic index of inhaled corticosteroids, UK371,104 was identified as a selective agonist of the adenosine A(2A) receptor that has a lung focus of pharmacological activity following delivery to the lung in a pre clinical in vivo model of lung function. Lung-focussed agents such as UK371,104 may be suitable for assessing the anti-inflammatory potential of inhaled adenosine A(2A) receptor agonists.     

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca²⁺ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK).
2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca²⁺, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca²⁺ had little effect on the peak Ca²⁺-response, but resulted in a more rapid decline to basal. There was no response to α,β-MethylATP (α,βMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage.
3. ATP (log EC₅₀ −5.1±0.2) also caused an increase in the total [³H]-inositol (poly)phosphates ([³H]-InsP_x) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and α,βMeATP giving no detectable response.
4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P₃.
5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPγS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 μM forskolin.
6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with activation at P2Y₂ receptors. 2MeSATP gave a much smaller response with a lower potency than UTP.
7. These results are consistent with brain endothelial regulation by P2Y₂ receptors coupled to phospholipase C, Ca²⁺ and MAPK; and by P2Y₁-like (2MeSATP-sensitive) receptors which are linked to Ca²⁺ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P₃ levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

     

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

Background and purpose:

P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.

Experimental approach:

mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE^–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE^–/– mice.

Key results:

P2Y₆ receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y₂ receptor expression remained unchanged. Expression of P2Y₁ or P2Y₄ receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y₆ mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y₆-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y₆ receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y₆-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE^–/– mice with suramin or PPADS (50 and 25 mg·kg⁻¹·day⁻¹ respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication.

Conclusions and implications:

These results suggest involvement of nucleotide receptors, particularly P2Y₆ receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y₆ receptor-deficient mice.     

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2′, 4′ disulphonic acid (PPADS) act as antagonists at transfected P2Y₁ receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors.
2. The expression of either P2Y₁ or P2Y₂ receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [³H]-inositol(poly)phosphates([³H]-InsP_x) compared to cells not expressing these receptors. This elevation is much greater in P2Y₁ transfectants than in P2Y₂ transfectants.
3. Both PPADS and suramin reduced this basal level of [³H]-InsP_x accumulation in the P2Y₁ expressing cells.
4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5′-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist.
5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [³H]-InsP_x accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y₁ expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.