Purinergic facilitation of ATP-sensitive potassium current in rat ventricular myocytes

1. The effects of different purinergic agonists on the cardiac adenosine 5′-triphosphate (ATP)-sensitive potassium current (I_K(ATP)), appearing during dialysis of rat isolated ventricular myocytes with a low-ATP (100 μM) internal solution under whole-cell patch-clamp conditions, were examined in the presence of a P₁ purinoceptor antagonist.
2. The extracellular application of ATP in the micromolar range induced, besides known inward currents through cationic and chloride channels, the facilitation of I_K(ATP) once I_K(ATP) had already been partially activated during the low-ATP dialysis.
3. Analogues of ATP, α,β-methyleneadenosine 5′-triphosphate (α,βmeATP), 2-methylthioadenosine triphosphate (2MeSATP), adenosine 5′-O-3-thiotriphosphate (ATPγS) similarly facilitated I_K(ATP). UTP and ADP were very weak agonists while AMP and adenosine had no detectable effect.
4. The half-maximal stimulating concentration (C₅₀) of α,βmeATP, an analogue that did not elicite the interfering inward cationic current was 1.5 μM. Similar apparent C₅₀ (1–2 μM) were observed for ATP and analogues tested with somewhat less maximal effect of ATPγS.
5. Suramin, a nonselective P₂-purinoceptor antagonist, altered I_K(ATP) at the relatively high concentration required to inhibit purinoceptors. Pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a supposedly predominantly P_2X-purinoceptor antagonist, at micromolar concentration inhibited the transient inward current but did not block the facilitation of I_K(ATP).
6. Our results demonstrate that ATP and its analogues facilitate I_K(ATP) in rat ventricular myocytes by stimulation of non-P₁-, non-P_2X-purinoceptors.

     

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.     

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.

     

Activity of diadenosine polyphosphates at P2Y receptors stably expressed in 1321N1 cells

The selectivities of the diadenosine polyphosphates (Ap_nAs, n=2–6) at the human P2Y₁, P2Y₂, P2Y₄, P2Y₆ and P2Y₁₁ receptors stably expressed in 1321N1 human astrocytoma cells was determined using a Fluorescence Imaging Plate Reader (FLIPR) to measure intracellular Ca²⁺ mobilisation. The rank order of agonist potencies at P2Y₁ were: ADP>P¹,P³-diadenosine triphosphate (Ap₃A)>P¹,P³-diadenosine hexaphosphate (Ap₆A)=P¹,P³-diadenosine diphosphate (Ap₂A)P¹,P³-diadenosine pentaphosphate (Ap₅A). P¹,P³-diadenosine tetraphosphate (Ap₄A) was inactive up to 1 mM. The rank order of agonist potencies at P2Y₂ were: UTP>Ap₄AAp₆A>Ap₅A>Ap₃AAp₂A. The Ap₄A concentration response curve appeared to be bi-phasic. At P2Y₄ all the Ap_nAs tested were inactive as agonists. At P2Y₆, only Ap₃A and Ap₅A showed significant agonist activity. At P2Y₁₁, only Ap₄A showed significant agonist activity. Ap_nAs were inactive as antagonists of the P2Y₁, P2Y₂, P2Y₄, P2Y₆ and P2Y₁₁ receptors. At P2Y₄, however, the Ap_nAs potentiated the UTP response.     

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.     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.

     

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.     

Effects of antagonists at the human recombinant P2X7 receptor

1. We have used whole-cell patch clamping methods to examine the properties of the recombinant human P2X₇ (P2Z) receptor stably expressed in HEK-293 cells.
2. In an extracellular solution with lowered concentrations of divalent cations (zero Mg²⁺ and 0.5 mM Ca²⁺), both ATP and the nucleotide analogue, 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (Bz-ATP) evoked concentration-dependent whole-cell inward currents with maxima of 4658±671 and 5385±990 pA, respectively, at a holding potential of −90 mV. Current-voltage relationships determined using 100 μM Bz-ATP reversed at −2.7±3.1 mV, and did not display significant rectification.
3. Repeated applications of 300 μM Bz-ATP produced inward currents with similar rise-times (approx. 450 ms, 5–95% current development) but with progressively slower 95–5% decay times, with the eighth application of this agonist yielding a decay time of 197% of the first application.
4. Concentration-effect curves to ATP and Bz-ATP produced estimated EC₅₀ values of 780 and 52.4 μM, respectively. Consecutive concentration-effect curves to Bz-ATP produced curves with similar maxima and EC₅₀ values.
5. The non-selective P2 antagonists, pyridoxal-phosphate-6-azophenyl-, 2′,4′-disulphonic acid (PPADS) and suramin, both produced concentration-dependent increases in maximal inward currents to Bz-ATP, with IC₅₀ concentrations of approximately 1 μM and 70 μM, respectively. The profile of antagonism produced by PPADS was not that of a competitive antagonist.
6. The isoquinolene derivatives 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62) and calmidazolium both produced antagonism which was not competitive, with IC₅₀ concentrations of approximately 15 and 100 nM, respectively. HMA (5-(N,N-hexamethylene)- amiloride) was also an effective antagonist at a concentration of 10 μM. The group IIb metal, copper, also displayed antagonist properties at the human P2X₇ receptor, reducing the maximum response to Bz-ATP by about 50% at a concentration of 1 μM.
7. These data demonstrate that the human recombinant P2X₇ receptor displays functional behaviour which is similar to the recombinant rat P2X₇ receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.

     

Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226

1. Experiments were conducted to evaluate the effects of the novel non-peptide neuropeptide Y Y₁ receptor antagonist, BIBP3226 (N²-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide) on spontaneous, fasting-induced and NPY-induced food intake in rats. In addition to consumption of regular chow, the effects of BIBP3226 on consumption of highly palatable sweetened mash were monitored in a 1 h test on first exposure and after familiarization with novel food.
2. BIBP3226 (10.0 nmol, i.c.v.) had no effect on the consumption of regular chow, but reduced significantly the intake of highly palatable diet and the food intake stimulated by fasting (24 h). Neuropeptide Y (NPY, 1.0 nmol, i.c.v.) significantly increased the consumption of regular rat chow. This orexigenic effect of NPY was blocked by BIBP3226 (10.0 nmol, administered i.c.v. 5 min before NPY) at 30  min and 4  h, but not at 1 and 2  h. When animals were pretreated with diazepam (0.5 mg kg⁻¹, i.p., 20 min before NPY), BIBP3226 failed to suppress NPY-induced feeding.
3. An NPY Y₁ and Y₃ receptor agonist, [Leu³¹,Pro³⁴]NPY and a Y₅ receptor agonist human peptide YY_3–36 (hPYY_3–36, both 30 pmol), microinjected into the paraventricular nucleus of the hypothalamus (PVN) increased the consumption of regular rat chow. BIBP3226 (0.4 nmol, into the PVN) completely blocked the stimulatory effect of [Leu³¹,Pro³⁴]NPY but not that of hPYY_3–36. BIBP3226 (0.4 nmol) alone failed to modify the consumption of the regular chow. Higher doses of BIBP3226 (1.0 and 2.0 nmol) injected into the vicinity of the PVN reduced the consumption of the sweetened mash.
4. These results suggest that both the NPY Y₁ and Y₅ receptors in the PVN are involved in the regulation of food intake. The stimulatory effect of exogenous NPY is probably mediated through an NPY receptor subtype that is not identical with the Y₁ receptor (possibly Y₅ receptor). However, the NPY Y₁ receptors may mediate the effect of endogenous NPY in conditions of increased energy demand or on intake of highly palatable diets.

     

Attenuation of morphine withdrawal symptoms by subtype-selective metabotropic glutamate receptor antagonists

1. We have previously shown that chronic antagonism of group I metabotropic glutamate receptors (mGluRs), in the brain, attenuates the precipitated morphine withdrawal syndrome in rats. In the present investigation we assessed the effects of chronic antagonism of group II and III mGluRs on the severity of withdrawal symptoms in rats treated chronically with subcutaneous (s.c.) morphine.
2. Concurrently with s.c. morphine we infused intracerebroventricularly (i.c.v.) one of a series of phenylglycine derivatives selective for specific mGluR subtypes. Group II mGluRs (mGluR_2,3), which are negatively coupled to adenosine 3′: 5′-cyclic monophosphate (cyclic AMP) production, were selectively antagonized with 2s, 1′s, 2′s-2-methyl-2-(2′-carboxycyclopropyl) glycine (MCCG). Group III mGluRs (mGluR_{4,6,7 and 8}), which are also negatively linked to cyclic AMP production, were selectively antagonized with α-methyl-L-amino-4-phosphonobutanoate (MAP4). The effects of MCCG and MAP4 were compared with α-methyl-4-carboxyphenylglycine (MCPG), which non-selectively antagonizes group II mGluRs, as well as group I mGluRs (mGluR_1,5) which are positively coupled to phosphatidylinositol (PI) hydrolysis.
3. Chronic i.c.v. administration of both MCCG and MAP4 significantly decreased the time spent in withdrawal, MCPG and MCCG reduced the frequency of jumps and wet dog shakes and attenuated the severity of agitation.
4. Acute i.c.v. injection of mGluR antagonists just before the precipitation of withdrawal failed to decrease the severity of abstinence symptoms. Rather, acute i.c.v. injection of MCCG significantly increased the time spent in withdrawal.
5. Our results suggest that the development of opioid dependence is affected by mGluR-mediated PI hydrolysis and mGluR-regulated cyclic AMP production.

     

Evidence for the involvement of P2-purinoceptors in the cholinergic contraction of the guinea-pig ileum

In the isolated ileum of the guinea-pig the P₂-purinoceptor antagonists PPADS and suramin: (a) strongly inhibited the cholinergic contractile effect of α,β-methylene ATP, (b) did not influence contractions evoked by exogenous acetylcholine (ACh) but, (c) moderately (by about 30%) inhibited cholinergic contractions due to electrical field stimulation (EFS), in a non-additive manner. These results suggest that an endogenous ligand that stimulates P₂-purinoceptors (possibly ATP) is involved in the contractile effect of EFS, as a positive modulator of ACh release.     

P2Y1 receptors mediate inhibitory neuromuscular transmission in the rat colon

Background and purpose:

Inhibitory junction potentials (IJP) are responsible for smooth muscle relaxation in the gastrointestinal tract. The aim of this study was to pharmacologically characterize the neurotransmitters [nitric oxide (NO) and adenosine triphosphate (ATP)] and receptors involved at the inhibitory neuromuscular junctions in the rat colon using newly available P2Y₁ antagonists.

Experimental approach:

Organ bath and microelectrode recordings were used to evaluate the effect of drugs on spontaneous mechanical activity and resting membrane potential. IJP and mechanical relaxation were studied using electrical field stimulation (EFS).

Key results:

N^ω-nitro-L-arginine (L-NNA) inhibited the slow component of the IJP and partially inhibited the mechanical relaxation induced by EFS. MRS2179, MRS2500 and MRS2279, all selective P2Y₁ receptor antagonists, inhibited the fast component of the IJP without having a major effect on the relaxation induced by EFS. The combination of both L-NNA and P2Y₁ antagonists inhibited the fast and the slow components of the IJP and completely blocked the mechanical relaxation induced by EFS. Sodium nitroprusside caused smooth muscle hyperpolarization and cessation of spontaneous motility that was prevented by oxadiazolo[4,3-α]quinoxalin-1-one. Adenosine 5′-O-2-thiodiphosphate, a preferential P2Y agonist, hyperpolarized smooth muscle cells and decreased spontaneous motility. This effect was inhibited by P2Y₁ antagonists.

Conclusions and implications:

The co-transmission process in the rat colon involves ATP and NO. P2Y₁ receptors mediate the fast IJP and NO the slow IJP. The rank order of potency of the P2Y₁ receptor antagonists is MRS2500 greater than MRS2279 greater than MRS2179. P2Y₁ receptors might be potential pharmacological targets for the regulation of gastrointestinal motility.     

Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurones and cloning of the rat Y2 receptor

1. Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y₂ receptor described here for the first time.
2. More than 70% of the hippocampal neurones were endowed with [¹²⁵I]-[Leu³¹,Pro³⁴]PYY Y₁-like receptor silver grain accumulations and Y₁ receptor immunostaining. These radio- and immuno-labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal-like neuronal cells, as confirmed by neurone-specific enolase and MAP-2 staining.
3. Competition binding profiles revealed that specific [¹²⁵I]-[Leu³¹,Pro³⁴]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y₁ receptor sub-type with [Leu³¹,Pro³⁴]substituted NPY/PYY analogues>>C-terminal fragments=pancreatic polypeptides, with the non-peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [¹²⁵I]-[Leu³¹,Pro³⁴]PYY of the newly cloned Y₄, Y₅ and Y₆ receptors.
4. The expression of the genuine Y₁ receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y₂-like/[¹²⁵I]-PYY_3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y₁ and the Y₂ receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero.
5. Taken together, these results suggest that the Y₁ receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.

     

Extracellular ATP activates ERK1/ERK2 via a metabotropic P2Y1 receptor in a Ca2+ independent manner in differentiated human skeletal muscle cells

ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca2+ transients in these cells via P2Y receptors which were not attributable to Ca2+ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC50 of 21.3 microM. The Ca2+ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca2+ pool was not involved in ATP triggered Ca2+ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Substantially, ATP activated IP formation via a P2Y1 receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y1 receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca2+ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP3 receptor mediated Ca2+ transients and Ca2+ insensitive phosphorylation of ERK1/2.     

P2-purinoceptor antagonists: III. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to suramin

Effects of suramin and five analogs or fragments of suramin were studied on contractions of the rat vas deferens elicited by ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. One compound, NF023, differed from suramin by removal of two p-methylbenzamido groups, whereas another, BSt101, differed from NF023 by additional removal of the three sulphonate residues from one of the terminal naphthalene rings.The compounds all shifted the concentration-response curve of ,-MeATP in the rat vas deferens to the right and simultaneously increased the maximum of the curve. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent K _d values were between 1 and 3672 M. In the guinea-pig taenia coli, the compounds shifted the concentration-response curve of ADPS to the right in a parallel manner, but in the one case where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent K _d values were between 10 and 786 M. The removal of ATP from the medium by vas deferens tissue was decreased only by suramin, NF023 and BSt101, with IC_25% values between 170 and 590 M.The results indicate that P_2X-purinoceptor affinity, P_2Y-purinoceptor affinity and the ecto-nucleotidase effect all increase with the size of the molecule. BSt101 resembled NF023 in potency at all three sites, indicating that the possession of a second naphthalene-trisulphonate group is not a prerequisite for relatively high affinity. NF023 is interesting because it is P_2X- versus P_2Y-selective and, in addition, the compound with the highest P_2X- versus ecto-nucleotidase-selectivity presently available.     

P2Y receptor specific for diadenosine tetraphosphate in lung: selective inhibition by suramin,PPADS, Ip5I,and not by MRS-2197

Extracellular dinucleotides, which act as signaling molecules in a variety of systems, may regulate fluid homeostasis in the human lung by activation of a specific P2Y receptor subtype. Previously, we presented evidence for a G protein-coupled P2Y receptor with high affinity for dinucleotides in both rat and human lung tissue. In a human bronchial epithelial cell line (HBE-1), diadenosine polyphosphates (Ap(n)A, n=2-6) increase intracellular Ca(2+). The aim of the present work was to find additional evidence that, in these cells, the receptors selectively activated by diadenosine polyphosphates are distinct from already known P2Y receptors, which are activated by the mononucleotides ATP or UTP. We tested antagonists suitable to classify P2Y receptor subtypes. The P2Y(1) receptor-selective antagonist 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS-2197) did not affect Ca(2+) mobilization induced by diadenosine tetraphosphate (Ap(4)A). However, suramin, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) and diinosine pentaphosphate (Ip(5)I) inhibited the Ca(2+) response by 96%, 92% and 32%, respectively. Moreover, these results were confirmed by assessing the specific binding of [3H]Ap(4)A to membranes from human and rat lung. Suramin (100 microM), PPADS (400 microM) and Ip(5)I (200 microM), reduced [3H]Ap(4)A binding in lung membrane preparations by 66%, 77% and 80%, respectively. The Ap(4)A-induced Ca(2+) response in HBE-1 cells was inhibited to a much greater extent by these antagonists than the ATP- or UTP-evoked Ca(2+) rise. Thus, Ap(4)A in lung epithelial cells also activates a still unidentified P2Y receptor that is specific for dinucleotides over mononucleotides.     

Acute nociception mediated by hindpaw P2X receptor activation in the rat

1. The functional consequences of P2X receptor activation on peripheral sensory neurones have been investigated in vivo. Behavioural indices of acute nociception were monitored in the conscious rat following subplantar injection of adenosine 5′-triphosphate (ATP), α,β-methylene ATP, adenosine 5′-diphosphate (ADP) and adenosine.
2. Signs of overt nociception, i.e. hindpaw lifting and licking, were apparent in animals injected subplantar with the P2X receptor agonist, α,β-methylene ATP. Nociceptive behaviours continued for 15 min following administration of α,β-methylene ATP (200 nmol) and were dose-related (0–5 min hindpaw lifting times after injection of α,β-methylene ATP 100 nmol and 1000 nmol were 89±26 s and 232±11 s, respectively). Subplantar ATP evoked a modest response only at the highest dose tested (1000 nmol; 0–5 min hindpaw lifting time 66±19 s) whilst ADP or adenosine (both 600 nmol) elicited negligible spontaneous nociceptive activity.
3. Morphine (3 mg kg⁻¹, i.v.) abolished hindpaw licking behaviour induced by subplantar injection of either α,β-methylene ATP (600 nmol) or bradykinin (1 nmol) and substantially reduced (88±5%) paw licking in formalin (0.5%, 0.1 ml) injected animals. In contrast, hindpaw lifting was only modestly inhibited (34±11%) in morphine-pretreated animals that had received subplantar bradykinin and was unaffected in rats in which the noxious stimulus was either subplantar α,β-methylene ATP or formalin. Pretreatment of hindpaws with subplantar bupivacaine (1% w/v, 0.1 ml) abolished α,β-methylene ATP-evoked nociceptive behaviours.
4. Hindpaw lifting and licking mediated by α,β-methylene ATP (600 nmol, subplantar) were inhibited (72±15% and 95±5%, respectively) by 30 min local pretreatment with 600 nmol α,β-methylene ATP. Subplantar α,β-methylene ATP pretreatment did not inhibit behaviour stimulated by subsequent bradykinin (1 nmol) or formalin (0.5%, 0.1 ml) injection into the hindpaw.
5. Desensitization of small diameter sensory neurones with a single subplantar injection of capsaicin (100 μg) abolished all behaviours indicative of spontaneous nociceptive sensation in animals subsequently injected with α,β-methylene ATP (600 nmol), bradykinin (1 nmol) or formalin (0.5%, 0.1 ml).
6. We conclude that activation of P2X receptors present on small diameter (capsaicin-sensitive) primary afferent neurones in the rat hindpaw mediates behaviour indicative of acute nociception.

     

Antiaggregatory activity in human platelets of potent antagonists of the P2Y 1 receptor

Activation of the P2Y(1) nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500(2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y(1) receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable CP bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC(50) value of 0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC(50) values of 62.8 nM and 1.5 microM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y(1) receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca(2+). No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y(1) receptor antagonists with the proaggregatory P2Y(12) receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y(1) receptor, and MRS2500 is the most potent such antagonist yet reported.