Search

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes divided into two subgroups (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) and (P2Y12, P2Y13, and P2Y14). The P2Y receptors are expressed in various cell types and play important roles in physiology and pathophysiology including inflammatory responses and neuropathic pain. The antagonism of P2Y12 receptors is used in pharmacotherapy for the prevention and therapy of cardiovascular events. The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel inhibit platelet P2Y12 receptors and reduce thereby platelet aggregation. The P2Y2 receptor agonist diquafosol is used for the treatment of the dry eye syndrome. The P2Y receptor subtypes differ in their amino acid sequences, their pharmacological profiles and their signaling transduction pathways. Recently, selective receptor ligands have been developed for all subtypes. The published crystal structures of the human P2Y1 and P2Y12 receptors as well as receptor models will facilitate the development of novel drugs for pharmacotherapy.

The Gi protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3′-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure–activity relationships (SARs) were steep. 3,3′-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC50 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further inve...

The orphan G protein-coupled receptor (GPCR) GPR84 is expressed on immune cells mediating proinflammatory and immunostimulatory effects. In this study, we prepared the fully efficacious, nonbiased GPR84 agonist 6-hexylamino-2,4(1H,3H)-pyrimidinedione (6) in tritium-labeled form ([3H]PSB-1584) by hydrogenation of a hexenyl-substituted precursor with tritium gas. The radioligand was characterized by kinetic, saturation, and competition assays using membranes of Chinese hamster ovary cells recombinantly expressing the human GPR84. [3H]6 reversibly labeled the receptor with high affinity (KD 2.08 nM). Structurally diverse orthosteric and allosteric ligands, including newly designed and synthesized compounds, were studied in competition binding assays. A homology model of GPR84 was generated to perform docking studies rationalizing the experimental data. The radioligand was additionally used for labeling GPR84 in native cells and tissues. [3H]6 constitutes the first GPR84 agonist radioligand representing a powerful tool for this poorly investigated GPCR, which has potential as a future drug target.

Professor Geoffrey Burnstock proposed the concept of purinergic signaling via P1 and P2 receptors. P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular adenine and uracil nucleotides. Eight mammalian P2Y receptor subtypes have been identified. They are divided into two subgroups (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) and (P2Y12, P2Y13, and P2Y14). P2Y receptors are found in almost all cells and mediate responses in physiology and pathophysiology including pain and inflammation. The antagonism of platelet P2Y12 receptors by cangrelor, ticagrelor or active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel reduces the ADP-induced platelet aggregation in patients with thrombotic complications of vascular diseases. The nucleotide agonist diquafosol acting at P2Y2 receptors is used for the treatment of the dry eye syndrome. Structural information obtained by crystallography of the human P2Y1 and P2Y12 receptor proteins, site-directed mutagenesis and molecular modeling will facilitate the rational design of novel selective drugs.

In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1β protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3 mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1β. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways., Highlights • Pharmacological blockade of P2Y12 receptors alleviates inflammatory and neuropathic pain. • Central inhibition of P2Y12 receptors attenuates cytokine production in the spinal cord. • Central P2Y12 receptor inhibition attenuates cytokine production in the inflamed hind paw. • α7-Receptors mediate the effect of P2Y12 receptor blockade on hyperalgesia and cytokine level. • Genetic deletion of P2Y12 receptors alleviates inflammatory, neuropathic and acute pain.

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation "P0-receptors" has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [(3)H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure-activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC50 9.77 nM) and the cAdeR (EC50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC50 6.24 nM) and was found to be additionally coupled to Gq proteins.

The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn194(5.40) and Leu201(5.47), were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.

The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25-Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.

The P2Y(12)-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y(12)-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular cAMP production and cAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced cAMP production with IC(50) concentrations of about 0.3nM. In cells expressing R256A constructs, the IC(50) concentration amounted to 25nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular cAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wild-type receptors and R256A constructs (apparent pK(B)-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent pK(B)-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y(12)-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor.

Hypotrichosis simplex is a group of nonsyndromic human alopecias. We mapped an autosomal recessive form of this disorder to chromosome 13q14.11-13q21.33, and identified homozygous truncating mutations in P2RY5, which encodes an orphan G protein-coupled receptor. Furthermore, we identified oleoyl-L-alpha-lysophosphatidic acid (LPA), a bioactive lipid, as a ligand for P2Y5 in reporter gene and radioligand binding experiments. Homology and studies of signaling transduction pathways suggest that P2Y5 is a member of a subgroup of LPA receptors, which also includes LPA4 and LPA5. Our study is the first to implicate a G protein-coupled receptor as essential for and specific to the maintenance of human hair growth. This finding may provide opportunities for new therapeutic approaches to the treatment of hair loss in humans.

The present study aimed at elucidating the molecular identity of the proposed “I 1 -imidazoline receptors”, i.e. non-adrenoceptor recognition sites via which the centrally acting imidazolines clonidine and moxonidine mediate a major part of their effects. In radioligand binding experiments with [ 3 H]clonidine and [ 3 H]lysophosphatidic acid on intact, α 2 -adrenoceptor-deficient PC12 cells, moxonidine, clonidine, lysophosphatidic acid and sphingosine-1-phosphate (S1P) competed for the specific binding sites of both radioligands with similar affinities. RNA interference with the rat S1P 1 -, S1P 2 - or S1P 3 -receptor abolished specific [ 3 H]lysophosphatidic acid binding. [ 3 H]Clonidine binding was markedly decreased by siRNA targeting S1P 1 - and S1P 3 -receptors but not by siRNA against S1P 2 -receptors. Finally, in HEK293 cells transiently expressing human S1P 3 -receptors, sphingosine-1-phosphate, clonidine and moxonidine induced increases in intracellular calcium concentration, moxonidine being more potent than clonidine; this is in agreement with the known properties of the “I 1 -imidazoline receptors”. The present results indicate that the “I 1 -imidazoline receptors” mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belong to the S1P-receptor family; in particular, the data obtained in PC12 cells suggest that the I 1 imidazoline receptors represent a mixture of S1P 1 - and S1P 3 -receptors and/or hetero-dimers of both.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). P2Y receptors are widely expressed and play important roles in physiology and pathophysiology. One important example is the ADP-induced platelet aggregation mediated by P2Y1 and P2Y12 receptors. Active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel as well as the nucleoside analogue ticagrelor block P2Y12 receptors and thereby platelet aggregation. These drugs are used for the prevention and therapy of cardiovascular events. Moreover, P2Y receptors play important roles in the nervous system. Adenine nucleotides modulate neuronal activity and neuronal fibre outgrowth by activation of P2Y1 receptors and control migration of microglia by P2Y12 receptors. UDP stimulates microglial phagocytosis through activation of P2Y6 receptors. There is evidence for a role for P2Y2 receptors in Alzheimer's disease pathology. The P2Y receptor subtypes are highly diverse in both their amino acid sequences and their pharmacological profiles. Selective receptor ligands have been developed for the pharmacological characterization of the receptor subtypes. The recently published three-dimensional crystal structures of the human P2Y1 and P2Y12 receptors will facilitate the development of therapeutic agents that selectively target P2Y receptors. This article is part of the Special Issue entitled ‘Purines in Neurodegeneration and Neuroregeneration’.

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Proc Natl Acad Sci USA 99:8573-8578, 2002). In the present study, we show for the first time that the adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Saturation analysis with [3H]adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of protein. Kinetic studies revealed unusual binding kinetics of [3H]adenine with rapid association and slow dissociation. A series of compounds were investigated in [3H]adenine competition experiments at rat brain cortex. Only minor substitution of the adenine structure was tolerated, the most potent compounds of the present series being 2-fluoroadenine (Ki value of 620 nM), 8-thioadenine (Ki value of 2.77 microM), N6-methyladenine (Ki value of 3.64 microM), and 7-methyladenine (Ki value of 4.13 microM), all of which were partial agonists (40-60% intrinsic activity). Adenine dose dependently inhibited forskolin-stimulated adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells. Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the adenine receptor. Moreover, preliminary radioligand binding studies with [3H]adenine at membranes of human astrocytoma 1321N1 cells suggest that a human ortholog of the rat adenine receptor exists.

The physiological role of the P2Y(6) nucleotide receptor may involve cardiovascular, immune and digestive functions based on the receptor tissue distribution, and selective antagonists for this receptor are lacking. We have synthesized a series of symmetric aryl diisothiocyanate derivatives and examined their ability to inhibit phospholipase C (PLC) activity induced by activation of five subtypes of recombinant P2Y receptors. Several derivatives were more potent at inhibiting action of UDP at both human and rat P2Y(6) receptors expressed in 1321N1 human astrocytes than activation of human P2Y(1), P2Y(2), P2Y(4) and P2Y(11) receptors. The inhibition by diisothiocyanate derivatives of 1,2-diphenylethane (MRS2567) and 1,4-di-(phenylthioureido) butane (MRS2578) was concentration-dependent and insurmountable, with IC(50) values of 126+/-15 nM and 37+/-16 nM (human) and 101+/-27 nM and 98+/-11 nM (rat), respectively. A derivative of 1,4-phenylendiisothiocyanate (MRS2575) inhibited only human but not rat P2Y(6) receptor activity. MRS2567 and MRS2578 at 10microM did not affect the UTP (100nM)-induced responses of cells expressing P2Y(2) and P2Y(4) receptors, nor did they affect the 2-methylthio-ADP (30nM)-induced responses at the P2Y(1) receptor or the ATP (10microM)-induced responses at the P2Y(11) receptor. Other antagonists displayed mixed selectivities. The selective antagonists MRS2567, MRS2575 and MRS2578 (1microM) completely blocked the protection by UDP of cells undergoing TNFalpha-induced apoptosis. Thus, we have identified potent, insurmountable antagonists of P2Y(6) receptors that are selective within the family of PLC-coupled P2Y receptors.

In the search for P2-receptors modulating the stimulation-evoked entry of calcium at processes of PC12 cells differentiated in the presence of nerve growth factor and neurotrophin-3, electrically evoked increases in free calcium were assessed by fura-2 microfluorimetry. Omission of calcium and addition of cadmium (100 microM) or the N-type calcium channel blocker omega-conotoxin GVIA (0.5 microM) abolished or markedly reduced the evoked responses. The P2Y-receptor agonists 2-methylthio adenosine 5'-diphosphate (2-methylthio-ADP), ADP, and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) inhibited the electrically evoked entry of calcium without any changes in basal calcium concentrations. 2-Methylthio-ADP was the most potent agonist. Adenosine, P(1),P(4)-di(adenosine-5')-tetraphosphate (Ap4A), UDP, and UTP (30 microM each) had no effect. The effect of ADPbetaS (30 microM) was abolished by the P2-antagonists reactive blue 2 (3 microM), suramin (100 microM), 2-methylthio-AMP (10 microM), p-chloromercuriphenyl sulfonic acid (1 microM), and AR-C 69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene adenosine 5'-triphosphate] (300 nM). In contrast, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (10 microM), the selective P2Y1-receptor antagonist MRS 2179 (N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate; 10 microM), as well as the adenosine A(1)-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 100 nM), caused no change. Pretreatment with pertussis toxin abolished the effect of ADPbetaS. Reverse transcriptase-polymerase chain reaction revealed the presence of mRNA for P2Y12-receptors in nondifferentiated and differentiated PC12 cells. The results indicate that processes of differentiated PC12 cells possess P2Y12-receptors coupling to pertussis toxin-sensitive G-proteins and mediating an inhibition of the stimulation-evoked entry of calcium through omega-conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.

Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors. So far, the P2Y family is composed of eight cloned and functionally defined subtypes. Five of them (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11) are present in human tissues. The P2Y3-, p2y8- and tp2y-receptors may be species orthologues. The principal physiological agonists of the cloned human P2Y-receptors are ADP (P2Y1), UTP/ATP (P2Y2), UTP (P2Y4), UDP (P2Y6) and ATP (P2Y11). The rat P2Y4-receptor is activated by both UTP and ATP. Specific patterns of polar amino acid residues in the exofacial portions of transmembrane domains (TMs) 6 and 7 of the P2Y-receptors may account for the ligand specificity of the subtypes. Suramin acts as an antagonist at most P2Y-receptors with the exception of P2Y4- and tp2y-receptors. PPADS has been shown to block P2Y1-, the human P2Y4- and P2Y6-receptors. The nucleotide analogue 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS 2179), in contrast, seems to be a potent and selective antagonist at the P2Y1-receptor. All cloned and functionally expressed P2Y-receptors are able to couple to phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase and the tp2y-receptor an inhibition of this signal transduction pathway. Other functionally defined subtypes, e.g., the receptor mediating an inhibition of adenylate cyclase in blood platelets, are not yet cloned. The distribution of P2Y1 mRNA is widespread. The receptor plays a crucial role in blood platelet aggregation and mediates the adenine nucleotide-induced release of the endothelium-derived relaxing factor nitric oxide. P2Y1-receptors may also be involved in the modulation of neuro-neural signalling transmission. P2Y2 transcripts are abundantly distributed. One important example for its functional role is the control of chloride ion fluxes in airway epithelia. The P2Y4-receptor is highly expressed in the placenta. The distribution of the P2Y6-receptor is widespread including heart, blood vessels and brain. The P2Y11-receptor may play a role in the differentiation of immunocytes.

Purinoceptors mediate renal vasodilation by nitric oxide dependent and independent mechanisms. Background Adenosine triphosphate (ATP) and its metabolites including adenosine modulate renal vascular tone under physiological and pathophysiological conditions. Their effects are brought about by activation of membrane bound P 1 - and P 2 -purinoceptors located on smooth muscle and endothelial cells. In this study we analyzed the purinoceptor mediated dilation of rabbit and human renal arteries, and evaluated the possible involvement of endothelium-derived relaxing factors. Methods Segments of rabbit and human renal arteries were incubated and perfused with medium containing indomethacin. After preconstriction, drug induced changes in the vessel diameters were measured by a photoelectric device. Results ATP (EC 50 = 1 μmol/liter), added intraluminally, caused maximal vasodilation of 80 to 100% of the preconstriction response in both species. This effect was inhibited by the P1-purinoceptor antagonist 8-p-(sulphophenyl)theophylline (100 μmol/liter), suggesting that it was in part due to breakdown of ATP to adenosine. The nature of purinoceptor mediated renal vasodilation was studied further in rabbit renal arteries. Adenosine (EC 50 = 1 μmol/liter) as well as the P2Y-receptor agonists ADPβS (EC 50 = 0.4 μmol/liter) and 2-MeSATP (EC 50 = 0.2 μmol/liter) dilated the arteries by 80 to 100%. The effects of 2-MeSATP, which were to a much lesser extent that of ADPβS but not that of adenosine, were attentuated by the P2Y-antagonist reactive blue 2 (3 μmol/liter). Removal of the endothelium almost abolished the vasodilation induced by adenosine and ATP. In contrast, these dilator responses were only slightly attenuated by the nitric oxide synthase blockers N G -nitro-L-arginine methyl ester and N G -nitro-L-arginine (300 μmol/liter each), whereas acetylcholine and 2-MeSATP induced dilation was markedly reduced by N G -nitro-L-arginine methyl ester. Conclusions P1-purinoceptors activated by adenosine dilate rabbit renal arteries by an endothelium-derived relaxing factor that appears to be distinct from nitric oxide. In contrast, P2Y-purinoceptor induced renal dilation is mediated by nitric oxide. ATP, the physiological activator of P2Y-purinoceptors, is rapidly broken down to adenosine in rabbit and human renal arteries. Therefore, in rabbit and human renal arteries the vasodilatory effect of exogenous ATP mainly results from P1-purinoceptor activation probably through its breakdown product, adenosine.

Activation of ionotropic but not metabotropic glutamate receptors causes an indirect inhibition of the release of noradrenaline in slices of rabbit brain cortex. The inhibition is mediated by adenosine which activates presynaptic adenosine A1-receptors. The present study characterizes the ionotropic receptor types through which glutamate itself produces this indirect inhibition. Rabbit brain cortex slices were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM) and stimulated electrically by trains of 6 pulses at 100 Hz. Glutamate (100-3000 micro M) reduced the electrically evoked overflow of tritium by up to 58%. The effect did not differ 20 min and 60 min after addition of glutamate. Adenosine deaminase (1 U ml-1) as well as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 30 microM) and D-gamma-glutamylamino-methanesulfonate (GAMS; 30 micro M), both of which block kainate receptors, attenuated the glutamate-induced inhibition. The NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5; 100 micro M) and the AMPA receptor antagonist 6-nitro-7-sulfamoylbenzo(f)-quinoxaline-2,3-dione (NBQX; 30 micro M) did not change the effect of glutamate. Given alone, CNQX and GAMS, but not AP5 and NBQX, slightly increased the evoked overflow of tritium; the increases were abolished in the presence of adenosine deaminase. The results indicate that activation of kainate but not NMDA and AMPA receptors is involved in the indirect, adenosine-mediated inhibition by exogenous glutamate of the release of noradrenaline in rabbit brain cortex slices. Moreover, as shown by the increase caused by CNQX and GAMS, endogenous excitatory amino acids inhibit the release of noradrenaline through the kainate receptor-adenosine mechanism and thus contribute to the purinergic inhibitory control of noradrenaline release in the brain.

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release. In tissues preincubated with [3H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 μM and even more so by prazosin 0.3 μM combined with suramin 300 μM. They were also reduced by nifedipine 10 μM and even more so by nifedipine 10 μM combined with ryanodine 20 μM (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [3H]-noradrenaline, exogenous noradrenaline 10 μM and α,β-methylene ATP 10 μM elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 μM but not suramin 300 μM and were greatly reduced by nifedipine 10 μM and in Ca2+-free medium. Responses to α,β-methylene ATP were blocked by suramin 300 μM but not prazosin 0.3 μM were reduced by nifedipine 10 μM (effect on ATP overflow not significant) and were reduced even more in Ca2+-free medium. Neuropeptide Y 0.3 μM caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium. Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both α1-adrenoceptor and P2-purinoceptor activation. Ca2+ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A “purely neural” overflow of ATP was not isolated under the conditions of the experiments.

The purinergic response of the guinea-pig vas deferens to long trains of pulses at high frequency consists of an initial twitch followed by a much lower plateau. Mechanical, neurochemical and electrophysiological techniques were used to examine the reason for the fade. Mechanical measurements. In tissues stimulated by trains of 180 pulses/10 Hz and treated with prazosin to suppress the noradrenergic contraction component, the response to alpha, beta-methylene ATP and to exogenous ATP was as high during the secondary plateau of the purinergic neurogenic contraction as it was outside electrical stimulation periods; the response to 50 pulses/100 Hz was also unchanged during the low plateau. The plateau was not increased by reactive blue 2,8-(p-sulphophenyl)theophylline, propranolol or capsaicin. Neurochemical measurements. In tissues preincubated with [3H]-noradrenaline, electrical stimulation elicited an overflow of tritium and of ATP. In the absence of drugs as well as in the presence of prazosin and suramin to suppress contractions, the overflow of tritium per pulse decreased slightly in the course of trains of 90 pulses/10 Hz; the overflow of ATP per pulse decreased to a greater extent on average, but the decrease was not statistically significant. In the presence of prazosin and nifedipine, also to suppress contractions, the overflow of tritium per pulse again decreased slightly in the course of trains of 105 pulses/10 Hz, but the overflow of ATP per pulse if anything tended to increase. Electrophysiological measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

The P2Y receptors are a widely expressed group of eight nucleotide-activated G protein-coupled receptors (GPCRs). The P2Y(1)(ADP), P2Y(2)(ATP/UTP), P2Y(4)(UTP), P2Y(6)(UDP), and P2Y(11)(ATP) receptors activate G(q) and therefore robustly promote inositol lipid signaling responses. The P2Y(12)(ADP), P2Y(13)(ADP), and P2Y(14)(UDP/UDP-glucose) receptors activate G(i) leading to inhibition of adenylyl cyclase and to Gβγ-mediated activation of a range of effector proteins including phosphoinositide 3-kinase-γ, inward rectifying K(+) (GIRK) channels, phospholipase C-β2 and -β3, and G protein-receptor kinases 2 and 3. A broad range of physiological responses occur downstream of activation of these receptors ranging from Cl(-) secretion by epithelia to aggregation of platelets to neurotransmission. Useful structural models of the P2Y receptors have evolved from extensive genetic analyses coupled with molecular modeling based on three-dimensional structures obtained for rhodopsin and several other GPCRs. Selective ligands have been synthesized for most of the P2Y receptors with the most prominent successes attained with highly selective agonist and antagonist molecules for the ADP-activated P2Y(1) and P2Y(12) receptors. The widely prescribed drug, clopidogrel, which results in irreversible blockade of the platelet P2Y(12) receptor, is the most important therapeutic agent that targets a P2Y receptor.

1. Glutamate inhibits the electrically evoked release of noradrenaline in rabbit brain cortex slices; the inhibition is mediated by adenyl compounds, presumably adenosine. The aim of the present study was to identify the receptors involved in this indirect inhibitory effect of glutamate. Slices of the occipitoparietal cortex were preincubated with [3H]-noradrenaline and then superfused and stimulated by trains of 6 pulses, 100 Hz. 2. The ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AM-PA; 10-100 microM), kainate (10-100 microM) and N-methyl-D-aspartate (NMDA; 30-300 microM) but not the metabotropic glutamate receptor agonist, 1-amino-1,3-cyclopentanedicarboxylate (ACPD; 10-100 microM) reduced the electrically evoked overflow of tritium. 3. The effects of AMPA, kainate and NMDA were attenuated or abolished by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine deaminase but not by the alpha 2-adrenoceptor antagonist yohimbine, the gamma-aminobutyric acid (GABA) receptor antagonists, bicuculline and 2-hydroxysaclofen and the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). 4. The NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5) blocked the inhibitory effect of NMDA but not that of AMPA and kainate. The non-NMDA-receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the effect of AMPA but not of kainate and NMDA. 5. In addition to decreasing the electrically evoked overflow of tritium, AMPA, kainate and NMDA but not ACPD caused a steep but transient rise of basal tritium efflux. This immediate releasing effect was not significantly changed by DPCPX, adenosine deaminase, yohimbine, bicuculline, 2-hydroxysaclofen and L-NAME (except that L-NAME enhanced the effect of kainate). AP5 and CNQX antagonized the immediate releasing effects in the same way that they antagonized the inhibition by AMPA, kainate and NMDA of the electrically evoked overflow of tritium.6. It is concluded that AMPA, kainate and NMDA, like glutamate, reduce the electrically evoked release of noradrenaline by releasing adenosine or an adenine nucleotide which is then degraded to adenosine. Activation of each of the three ionotropic glutamate receptors, AMPA, kainate and NMDA receptors, but not activation of metabotropic glutamate receptors can initiate this indirect inhibitory effect on the release of noradrenaline (as well as the known noradrenaline releasing effect).

Contractions, release of previously stored [3H]-noradrenaline (measured as overflow of total tritiated compounds) and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) were studied in the superfused vas deferens of the guinea pig. Prazosin and suramin were used to suppress non-neural ATP release, and effects of bromoxidine and rauwolscine on the neural release thus isolated were examined. Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. Both prazosin (0.03-3 microM) and suramin (30-300 microM) reduced contractions as well as the evoked overflow of ATP. No visible contraction remained in 21 of 28 tissues exposed to prazosin 0.3 microM combined with suramin 300 microM. The evoked overflow of ATP under these conditions was about 17% of that observed in the absence of drugs. In the presence of prazosin 0.3 microM and suramin 300 microM, bromoxidine (0.01-1 microM) decreased and rauwolscine (0.1-10 microM) increased the evoked overflow of both tritium and ATP. Rauwolscine increased the evoked overflow of tritium to a significantly greater extent than the overflow of ATP. It is concluded that the overflow of ATP elicited by electrical (neural) stimulation in the presence of prazosin 0.3 microM and suramin 300 microM reflects purely neural release of ATP. This release of ATP, like the release of noradrenaline, is modulated through prejunctional alpha 2-adrenoceptors. The alpha 2-adrenoceptor modulation of the release of noradrenaline seems to be more marked than the modulation of the release of ATP.

1. The effects of nifedipine and ryanodine on the adrenergic component of neurogenic contractions of the rat isolated vas deferens were studied in an attempt to identify the sources of Ca2+ mediating the contraction. The tissue was electrically stimulated by single pulses or pairs of widely spaced pulses. The purinergic component of contraction was suppressed by the presence of 300 microM suramin. 2. In Mg(2+)-free medium, nifedipine (0.01-10 microM) reduced the first and, to a greater extent, the second twitch elicited by two pulses 3 s apart. This pattern of inhibition was observed both in the absence of rauwolscine (when twitch 2 was smaller than twitch 1) and in the presence of 0.1 microM rauwolscine (when, due to interruption of prejunctional alpha 2-adrenoceptor-mediated autoinhibition, twitch 2 was of similar height to twitch 1). Nifedipine reduced only twitch 2 but not twitch 1 in medium containing 1.2 mM Mg2+. 3. Single pulses of increasing current strength elicited increasing contraction. Nifedipine reduced contractions by about the same absolute extent at all current strengths, so that the relative contribution of the nifedipine-sensitive component decreased with increasing current strength. 4. When the pulse interval in a pair was increased from 5 to 60 s, the inhibition by nifedipine of the second twitch was most marked at an interval of 5 s and declined as the interval increased. 5. In contrast to nifedipine, 20 microM ryanodine reduced the first twitch of a pair to a greater extent than a second twitch 5 s later, so that twitch 2 became greater than twitch 1. The inhibition by ryanodine of twitch 2 increased with increasing pulse interval.6. In vasa deferentia preincubated with [3H]-noradrenaline, I microM nifedipine and 20 microM ryanodine did not change the electrically evoked overflow of tritium, whereas 10 microM nifedipine increased it.7. It is concluded that, when the sympathetic axons of the vas deferens are stimulated by a single pulse(or the first pulse of a pair) in Mg2+-free medium, both Ca2+ mobilization inside the smooth muscle cells and Ca2+ entry contribute to the ensuing adrenergic contraction. The relative contribution of Ca2+ entry is small at maximal stimulus strength but increases with decreasing stimulus strength. When a second pulse follows the first after an appropriate interval, a switch of Ca2+ sources occurs: intracellular Ca2+mobilization is decreased during twitch 2, whereas Ca2+ entry is increased.

1. Effects of opioid agonists on the purinergic and adrenergic components of neurogenic contractions and in some experiments on transmitter overflow were studied in the mouse isolated vas deferens. 2. When the vas deferens was stimulated every 2 min by pairs of pulses 2 s apart in the presence of prazosin 0.3 microM (to isolate the purinergic component) or alpha,beta-methylene-ATP 3 microM (to isolate the adrenergic component), each pulse elicited a separate twitch. The opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO, mu-receptor-selective), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-selective) and trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide (U-50488, kappa-selective) concentration-dependently reduced both purinergic and adrenergic contractions. For each agonist, maximal effects and concentrations causing half-maximal effects were very similar for inhibition of the purinergic component on the one hand and for inhibition of the adrenergic component on the other hand, although the adrenergic component was inhibited with a slight preference. Moreover, effects on contractions elicited by the first and the second pulse of the pairs were very similar. 3. When vasa deferentia preincubated with [3H]-noradrenaline were stimulated with trains of 100 pulses delivered at 20 Hz, morphine 10 microM reduced significantly both evoked tritium overflow and evoked contractions. Its effect was antagonized by naloxone. 4. It is concluded that, in contrast to drugs acting at some other presynaptic receptors, opioid mu-, delta- and kappa-agonists inhibit purinergic and adrenergic neurogenic contractions of the mouse vas deferens in a similar manner. In contrast to a previous report, no enhancement by morphine of the release of noradrenaline elicited by high frequency pulse trains was observed.

The P2Y(12) receptor plays a crucial role in platelet aggregation. In the present study, we analyzed the properties of non-nucleotide antagonists at the recombinant human P2Y(12) receptor and searched for amino acids involved in the molecular interaction. Receptor function was assessed by measuring the cAMP response element (CRE)-directed luciferase expression in Chinese hamster ovary cells. The cellular cAMP production was accelerated by forskolin; 2-methylthio-ADP was used to activate the wild-type P2Y(12) receptor or mutant constructs. 2-Methylthio-ADP inhibited the CRE-dependent luciferase expression with an IC(50) value of approximately 1 nM. The anthraquinone derivative reactive blue 2 used at increasing concentrations shifted the concentration-response curve of 2-methylthio-ADP to the right in a manner compatible with competitive antagonism (pA(2) value, 7.4). Its analog, 1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-0739), showed a markedly higher antagonistic potency with a pA(2) value of 9.8. In cells expressing the R256A-mutant receptor, the potencies of both reactive blue 2 (apparent pK(B), 5.9) and PSB-0739 (apparent pK(B), 9.1) were decreased. The same was true for the pure reactive blue 2 meta- and para-isomers and for the ortho-isomer cibacron blue 3GA. In contrast, the analog, 1-amino-4-[4-anilino-phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, lacking a sulfonic acid residue at ring D (PSB-0826), showed similar pK(B) values at wild-type (8.4) and R256A-mutant receptors (8.3). In summary, the results demonstrate that PSB-0739 is the most potent competitive non-nucleotide antagonist at the human P2Y(12) receptor described so far. The results also indicate that the sulfonic acid residue at ring D is involved in the interaction of antagonists derived from reactive blue 2 with the residue Arg256 of the human P2Y(12) receptor.

Hypotrichosis simplex (HS) is a group of isolated alopecias that can be inherited as an autosomal-dominant or an autosomal-recessive trait. Hair loss usually begins in early childhood, and is diffuse and progressive. Mutations in LIPH, which encodes lipase member H, have recently been shown to cause an autosomal-recessive form of HS. Here we describe an Austrian HS patient who was found to be carrying compound heterozygous mutations in the LIPH gene: a 7-bp frameshift duplication (c.403_409dup; p.Gln137HisfsX1) and a recently reported 30-amino acid in-frame duplication (c.280_369dup; p.Gly94_Lys123dup). To examine the impact of LIPH mutations on lipid metabolism, we established an in vitro assay to measure the action of this phospholipase in a cell-based system. Both the 7-bp duplication frameshift mutation and all known in-frame mutations were observed to reduce the in vitro activity of the lipase in response to the addition of phosphatidic acid, the substrate of lipase H. The reduced production of lysophosphatidic acid (LPA) led to a reduced response of cells expressing the human G-protein-coupled receptor p2y5 (p2y5) receptor. Our study increases the spectrum of known LIPH mutations and provides biochemical evidence for the important role of lipase H and its product LPA in human hair growth.

P2Y(12) plays an important role in platelet aggregation, which makes it an interesting target for antithrombotic agents. Compounds that antagonize P2Y(12) include the active metabolites of thienopyridines and molecules that are structurally related to ATP, which is an antagonist of P2Y(12). During the last few years, our group has been working on the development of P2Y(12) receptors antagonists that are based on an extremely simple chemical structure, the 6-amino-2-mercapto-3H-pyrimidin-4-one, variously substituted at the sulfur and oxygen functions. This nucleus represents the simplified combination of two known P2Y(12) antagonists: the active metabolite of the thienopyridines and ATP derivatives. The effects of the synthesized compounds were tested on ADP-induced human platelet aggregation, using light transmission aggregometry. None of the tested compounds induced platelet aggregation, while some of them, at concentration of 10(-4)M, partially inhibited platelet aggregation induced by ADP 10(-6)M. The most potent compound, 6b, antagonized the inhibitory effect of 2-methylthio-ADP on the forskolin-induced accumulation of cyclic-AMP in CHO FlpIN cells expressing recombinant human P2Y(12)-receptors. In addition, none of the tested compounds, including 6b, interfered with ligand binding to P1 receptors. Our results suggest that some of the synthesized compounds are specific antagonists of P2 receptors, and in particular of P2Y(12) and suggest that further development of this structurally new series of compounds as P2Y(12) receptors antagonists is recommended.

An orphan G protein-coupled receptor from the rat has recently been demonstrated to act as a transmembrane receptor for the nucleobase adenine. The receptor is possibly involved in nociception. Here we report the cloning and functional expression of an additional Gi-coupled receptor for adenine (Genbank accession code DQ386867). mRNA for this receptor was obtained from mouse brain and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. The new mouse protein sequence shares only 76% identity with that of the rat adenine receptor, suggesting that the receptors are not species homologs but distinct receptor subtypes. In human 1321N1 astrocytoma cells stably expressing the new mouse receptor, adenine and 2-fluoroadenine inhibited the isoproterenol-induced cAMP formation with IC50 concentrations of 8 and 15 nM, respectively. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM) as well as the P2 receptor antagonist suramin (300 μM) failed to change the responses to adenine. In contrast, pretreatment of cells with pertussis toxin abolished the effect of adenine. When the novel adenine receptor was expressed in Sf21 insect cells, a specific binding site for [3H]adenine was detected. In competition assays, the rank order of potency of selected ligands was identical to that obtained in membranes from NG108-15 cells and rat brain cortex (adenine > 2-fluoroadenine > 7-methyladenine > 1-methyladenine ≫ N6-dimethyladenine). In summary, our data show that a second mammalian DNA sequence encodes for a Gi-coupled GPCR activated by low, nanomolar concentrations of adenine.

The present study aimed at investigating the influence of imidazoline receptor ligands on the proliferation of PC12 cells and the involvement of the sphingosine-1-phosphate (S1P) signaling system in this effect. In cultured PC12 cells, S1P (0.3-100 nM) and the I(1) imidazoline receptor ligands moxonidine (0.3 and 1 mM), agmatine (1 mM), idazoxan (10-100 microM) and efaroxan (1-100 microM) concentration-dependently reduced protein contents which were used as estimates for cell number. The antiproliferative effects elicited by the compounds were abolished after knock-down of S1P(1), S1P(2) and S1P(3) receptors by RNA interference indicating an involvement of S1P receptors. In conclusion, the present data add further evidence to the recent finding that effects of imidazoline receptor ligands in PC12 cells are mediated by homo- and heterodimers of members of the S1P receptor family.

1. This study investigated a local effect of cooling on the plantar skin blood flow (PSBF) of tetrodotoxin-treated rats by laser-Doppler flowmetry. 2. When the air temperature around the left foot was locally cooled from 25 to 10 degrees C, the PSBF of the left foot decreased. 3. The response was inhibited by the alpha-adrenoceptor antagonist phentolamine, the alpha1-adrenoceptor antagonist bunazosin, the alpha2-adrenoceptor antagonist RS79948, and bretylium and guanethidine that inhibit noradrenaline release from sympathetic nerves. Adrenalectomy of the rats did not affect the cooling-induced response. 4. The P2 purinoceptor antagonists suramin and PPADS also significantly suppressed the cooling-induced reduction of PSBF. However, the inhibitory effect of PPADS on the cooling-induced response was abolished after the treatment with phentolamine. Intra-arterial injections of ATPgammaS, a stable P2 purinoceptor agonist, at 25 degrees C caused a transient decrease in PSBF in a dose-dependent manner, which was significantly inhibited by phentolamine and guanethidine. 5. These results suggest a novel mechanism for local cooling-induced reduction of skin blood flow in vivo; moderate cooling of the skin induces the release of ATP, which stimulates presynaptic P2 purinoceptors on sympathetic nerve terminals and facilitates the release of noradrenaline, thereby causing contractions of skin blood vessels via the activation of alpha1-and alpha2-adrenoceptors.

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.

The present study investigates the pharmacological profile of P2-purinoceptors modulating noradrenaline release from cultured chick sympathetic neurons. ATP (30 μM-3 mM) and 2-methylthio-ATP (3–100 μM), but not α,β-methylene-ATP (up to 100 μM), caused a significant facilitation of electrically evoked [3H]-noradrenaline release when added 2 min before depolarization. The facilitation declined with time of exposure suggesting receptor desensitization. The facilitatory effect was markedly diminished by the P2-purinoceptor antagonists reactive blue 2 (3 μM) and suramin (300 μM), but not changed by mecamylamine (10 μM), a nicotinic receptor antagonist. At 1 mM and higher concentrations, ATP added for 12 min, inhibited noradrenaline release; release was virtually abolished by 6 mM ATP. The inhibitory effect of ATP was slightly diminished by suramin but not affected by reactive blue 2. Electrically evoked [3H]-noradrenaline release remained unaffected in the presence of the adenosine (P1)-receptor agonists R(−)N6-(2-phenylisopropyl)adenosine (R-PIA), 2-[p-(2-carboxyethyl)phenylethylamino]5′-N-ethylcarboxamidoadenosine (CGS-21680), 5′-N-ethylcarboxamidoadenosine (NECA), and N6-2-(4-aminophenyl)ethyladenosine (APNEA), used up to 1 μM. The present results confirm the existence of two P2-purinoceptors affecting noradrenaline release: 1) a facilitatory receptor which is activated by 2-methyl thio-ATP as well as ATP, and blocked by suramin as well as reactive blue 2, and 2) an inhibitory receptor which is activated by ATP, only slightly affected by suramin but not at all by reactive blue 2 and does not belong to the established P2-purinoceptor subtypes.

Abstract The present study investigates sympathetic cotransmission and its α-adrenoceptor–mediated modulation in kidneys of spontaneously hypertensive rats (SHR, 12 to 14 weeks) and age-matched normotensive Wistar-Kyoto rats (WKY). In the presence of cocaine and corticosterone, renal nerve stimulation at 1 Hz (30 seconds) induced a greater outflow of norepinephrine in SHR (4.2±0.2 pmol/g kidney) than in WKY (3.0±0.2 pmol/g kidney). The α 2 -adrenoceptor antagonist rauwolscine (0.01 to 1 μmol/L) increased the stimulation-induced norepinephrine outflow to a greater extent in SHR than in WKY. In contrast, the α 1 -adrenoceptor antagonist prazosin (0.03 to 3 μmol/L) increased the stimulation-induced norepinephrine outflow to a greater extent in WKY than in SHR. This difference was not observed in the presence of the P 1 -purinoceptor antagonist 8-( p -sulfophenyl)theophylline (100 μmol/L). Stimulation at 4 Hz (30 seconds) induced an outflow of ATP (SHR, 12.7±3.3 pmol/g kidney; WKY, 16.7±2.1 pmol/g kidney; perfusion solution without cocaine and corticosterone). Prazosin (0.03 μmol/L) markedly reduced pressor responses to stimulation and inhibited the induced ATP outflow by 60% to 70%. When prazosin (0.03 μmol/L) was present, rauwolscine (0.1 μmol/L) increased the induced outflow of norepinephrine and ATP and markedly enhanced prazosin-resistant pressor responses. These pressor responses were abolished by the P 2 -purinoceptor antagonist suramin (300 μmol/L). The results demonstrate an increased α 2 -adrenoceptor–mediated automodulation of norepinephrine release in SHR kidneys caused by increased intrasynaptic norepinephrine levels. α 1 -Adrenoceptor–mediated transjunctional modulation of norepinephrine release by endogenous adenosine is defective in SHR kidneys and may be responsible for the greater norepinephrine release in this strain. Norepinephrine and ATP are coreleased in SHR and WKY kidneys and both mediate pressor responses to stimulation. The release of ATP is identical in SHR and WKY and is, like that of norepinephrine, modulated by α 2 -adrenoceptor–mediated autoinhibition.

1. We looked for P2-purinoceptors modulating noradrenaline release in rat heart atria. Segments of the atria were preincubated with [3H]-noradrenaline and then superfused with medium containing desipramine (1 microM) and yohimbine (1 microM) and stimulated electrically, by 30 pulses/1 Hz unless stated otherwise. 2. The adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; EC50 9.7 nM) and the nucleotides, ATP (EC50 6.6 microM) and adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; EC50 4.8 microM), decreased the evoked overflow of tritium. The adenosine A2a-agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.03-0.3 microM) and the P2x-purinoceptor agonist beta, gamma-methylene-L-ATP (30 microM) caused no change. 3. The concentration-response curve of CPA was shifted to the right by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX; 3 nM; apparent pKB value 9.7) but hardly affected by the P2-purinoceptor antagonist, cibacron blue 3GA (30 microM). In contrast, the concentration-response curves of ATP and ATP gamma S were shifted to the right by DPCPX (3 nM; apparent pKB values 9.3 and 9.4, respectively) as well as by cibacron blue 3GA (30 microM; apparent pKB values 5.0 and 5.1, respectively). Combined administration of DPCPX and cibacron blue 3GA caused a much greater shift of the concentration-response curve of ATP than either antagonist alone. The concentration-response curve of ATP was not changed by indomethacin, atropine or the 5'-nucleotidase blocker alpha, beta-methylene-ADP. 4. Cibacron blue 3GA (30 microM) increased the evoked overflow of tritium by about 70%. The increase was smaller when the slices were stimulated by 9 pulses/O00 Hz instead of 30 pulses/I Hz.5. The results indicate that the postganglionic sympathetic axons in rat atria possess P2-purinoceptors in addition to the known adenosine Al-receptor. Both mediate inhibition of noradrenaline release. Some adenine nucleotides such as ATP and ATP gamma S act at both receptors. The presynaptic P2-purinoceptor seems to be activated by an endogenous ligand, presumably ATP, under the condition of these experiments. This is the first evidence for presynaptic P2-purinoceptors at cardiac postganglionic sympathetic axons.

A new type of ligand for the study of P2-purinergic receptor subtypes was synthesized by combining and modifying conventional nucleoside chemistry with Fmoc solid phase peptide synthesis techniques. The tri- and tetra-aspartic acid derivatives of adenosine-5'-carboxylic acid (AdoCAsp3 and AdoCAsp4) were found to act as weak agonists at P2-purinergic receptors, (activated by ATP and UTP respectively) present on C6 glioma cells. AdoCAsp4 induced inositol 1,4,5-trisphosphate formation in the C6 cells with an EC50 of 73 microM. In addition, AdoCAsp4 was found to inhibit (IC50 approximately 80 microM) ATP-induced cytosolic [Ca2+] transients in these glioma cells. The glycine derivative, AdoCGly, increased evoked release of noradrenaline from mouse vas deferens slices, probably due to the blockade of presynaptic P2-autoreceptors. The possibility that aspartic, glutamic or gamma-carboxyglutamic residues may be used to replace phosphate groups on an ATP receptor ligand, opens up new ways in ligand design.

Contractions and overflow of tritium and ATP elicited by single electrical pulses or short pulse trains were studied in the guinea-pig isolated vas deferens preincubated with [3H]-noradrenaline. ATP was measured using the luciferase technique. A single pulse caused only a small contraction and minimal tritium and ATP overflow. In contrast, trains of 6 pulses elicited marked contractions as well as tritium and ATP overflow. In experiments with 6 pulses/100 Hz, prazosin 0.3 μM reduced the contraction by 73 %, did not change the evoked overflow of tritium, and reduced the evoked overflow of ATP by 85%. Suramin 300 μM reduced the contraction by 69% but changed neither the evoked overflow of tritium nor that of ATP. The combination of prazosin 0.3 gM and suramin 300 μM abolished the contraction, did not change the evoked overflow of tritium, and reduced the evoked overflow of ATP by 70%. When 6 pulses were applied at frequencies of 1, 2, 10 or 100 Hz, all responses increased with frequency up to a maximum at 10 Hz, but contractions and the evoked overflow of ATP increased with frequency to a greater extent than the evoked overflow of tritium. A similar frequency overflow relationship was observed when the medium contained prazosin 0.3 μM and suramin 300 μM (and evoked ATP overflow was greatly reduced). Yohimbine 1 μM did not affect the overflow of tritium evoked by 6 pulses/100 Hz but increased that evoked by 6 pulses/10 Hz. The results demonstrate an overflow of both noradrenaline and ATP in response to short pulse trains. As observed previously for prolonged pulse trains, the major part of the evoked overflow of ATP was derived from non-neural cells. The ATP overflow remaining during α1-adrenoceptor blockade by prazosin and P2-purinoceptor blockade by suramin is likely to reflect neural release of ATP. The results support the view that release of ATP increases with frequency to a greater extent than release of noradrenaline. The latency for the onset of prejunctional α2-autoinhibition in guinea-pig vas deferens is between 50 and 500 ms.

Effects of drugs acting at P2-purinoceptors on the release of newly taken up [3H]-noradrenaline were studied in slices of mouse and rat vas deferens. The slices were superfused and stimulated electrically, in most experiments by trains of 60 pulses/8 Hz. In mouse vas deferens, the P2-purinoceptor antagonists reactive blue 2 (1.8-100 microM) and brilliant blue G (10-300 microM) increased the stimulation-evoked overflow of tritium in a concentration-dependent manner as shown previously for suramin. Reactive blue 2, which preferentially blocks the P2Y-subtype, was the most potent compound and the compound with highest maximal effect, an increase by 104%. Pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in contrast, caused a small increase only at a single concentration (30 microM). The effects of reactive blue 2, brilliant blue G and suramin were not additive. The P2 agonist adenosine 5'-O-(3-thio)-triphosphate (ATP gamma S) reduced the evoked overflow of tritium. As shown previously for suramin, reactive blue 2 30 microM and brilliant blue G 100 microM antagonized the effect of ATP gamma S. From the shift of the ATP gamma S concentration-response curve to the right, an apparent pKB value of 5.3 was estimated for reactive blue 2 and an apparent pKB of 4.5 for brilliant blue G. In rat vas deferens, reactive blue 2 (3-30 microM), brilliant blue G (10 microM) and suramin (30-300 microM) also increased the evoked overflow of tritium. As in the mouse, reactive blue 2 was the most potent compound and the compound with highest maximal effect, an increase by 90%.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Prejunctional purinoceptors modulating the release of noradrenaline were compared in mouse and rat vas deferens. Tissue slices were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 60 pulses, 1 Hz. 2. In mouse vas deferens, 2-chloroadenosine (IC50 0.24 microM), beta,gamma-methylene-ATP (IC50 3.8 microM), alpha,beta-methylene-ATP (IC50 2.9 microM) and 2-methylthio-ATP (only 30 microM tested) reduced the evoked overflow of tritium. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), 10 nM, antagonized the effect of 2-chloro-adenosine (apparent pKB 10.2) as well as of beta,gamma-methylene-ATP (apparent pKB 9.6) and alpha,beta-methylene-ATP. Suramin, 300 microM, attenuated the effect of 2-chloroadenosine at best very slightly, antagonized the effect of beta,gamma-methylene-ATP (apparent pKB 4.5) and, when combined with DPCPX 10 nM, caused a further marked shift to the right of the concentration-response curve of beta,gamma-methylene-ATP beyond the shift produced by DPCPX alone. 3. In rat vas deferens, 2-chloroadenosine (IC50 0.20 microM), beta,gamma-methylene-ATP (IC50 4.8 microM), alpha,beta-methylene-ATP (IC50 3.0 microM) and 2-methylthio-ATP (only 30 microM tested) also reduced the evoked overflow of tritium. DPCPX, 10 nM, antagonized the effect of 2-chloroadenosine (apparent pKB 9.7) as well as of beta,gamma-methylene-ATP (apparent pKB 9.6) and alpha,beta-methylene-ATP. Suramin, 300 microM, did not change the effect of 2-chloroadenosine, attenuated the effect of beta,gamma-methylene-ATP at best very slightly and, when combined with DPCPX, caused at best a very small shift to the right of the concentration-response curve of beta,gamma-methylene-ATP beyond the shift produced by DPCPX alone.4. It is concluded that prejunctional purinoceptor mechanisms in mouse and rat vas deferens are similar. In either species, both nucleosides such as adenosine and nucleotides such as beta,gamma-methylene-ATP activate a common release-inhibiting receptor which is a Pl- or, more specifically, A1-purinoceptor.There seems to be no need to postulate the existence of a novel prejunctional P3-purinoceptor.Moreover, the sympathetic terminal axons possess an additional P2-purinoceptor in both species which is activated by some nucleotides such as beta,gamma-methylene-ATP and 2-methylthio-ATP, although the activation of the P2-purinoceptor by beta,gamma-methylene-ATP is difficult to demonstrate in the rat.

A possible contribution of adenine nucleotides to the endogenous purinergic, A1-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1–100 nmol/l) as well as the enzyme adenosine deaminase (0.1–10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The α1-adrenoceptor-selective agonist methoxamine (10 but not 1 μmol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 μmol/l but then not attenuated further by DPCPX100 nmol/l.L-Glutamate (300 μmol/l–2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 μmol/l but greatly, and to the same extent, attenuated by DPCPX 100 μmol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg++ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 μmol/l and 1 μmol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg++). It is concluded that adenosine but not an adenine nucleotide mediates the tonic purinergic presynaptic inhibition of noradrenaline release in rabbit brain cortex. The marked degree of disinhibition by DPCPX and adenosine deaminase underscores the potential physiological role of this inhibition. The purinergic inhibitory tone is reinforced by glutamate, indicating that glutamate releases adenyl compounds in rabbit brain cortex. Again adenosine but not an adenine nucleotide mediates the indirect inhibition by glutamate of the release of noradrenaline. The noradrenaline-releasing effect that glutamate exerts in rat occipito-parietal cortex does not occur in rabbit occipito-parietal cortex. Methoxamine depresses the release of noradrenaline in rabbit brain cortex directly at presynaptic α2-adrenoceptors rather than by release of purines.

Effects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz. Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADP beta S, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine greater than ATP approximately ATP gamma S approximately beta,gamma-imido-ATP approximately ADP greater than beta,gamma-methylene-ATP. AMP 30 mumol/l and AMPS 30 mumol/l were approximately equieffective with 30 mumol/l of adenosine and ATP gamma S, and ADP beta S 30 mumol/l was approximately equieffective with 30 mumol/l of ADP. alpha,beta-Methylene-ADP, 2-methylthio-ATP, UTP and GTP gamma S did not change the evoked overflow of tritium. alpha,beta-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to alpha,beta-methylene-ATP or when the slices were stimulated by 30 pulses, 10 Hz. Neither adenosine deaminase (100 U/l) nor the blocker of 5'-nucleotidase, alpha,beta-methylene-ADP (10 mumol/l), attenuated the inhibition caused by ATP, ATP gamma S and beta,gamma-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP to the right by very similar degrees. 8-(p-Sulphophenyl)-theophylline (30 and 300 mumol/l) also markedly antagonized the inhibition produced by ATP gamma S. alpha,beta-Methylene-ATP (10 and 30 mumol/l) and suramin (100 mumol/l) did not modify the effects of adenosine, ATP gamma S and beta,gamma-methylene-ATP. It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A1 subtype of the P1-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP can be potent P1 agonists. No evidence was found for presynaptic P2x-, P2y- or P3-purinoceptors.

Uridine 5′-triphosphate- (UTP-) and adenosine 5′-triphosphate-(ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure. Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5′-O-(3-thio)triphosphate (ATPγS), and β,γ-imido adenosine 5′-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, α, β-methylene-ATP and β,γ-methylene-ATP up to 10−3 mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP ≫ UMP = CTP; that of the purine nucleotides was: ATPγS > AMP-PNP > ATP > ADP > 2-methylthio-ATP = α, β-methylene-ATP = β,γ-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indometacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to α,β-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATPγS elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction. ATP 10−3 mol/l elicited marked additional vasoconstriction after precontraction with UTP 10−3 mol/l, whereas UTP elicited only a very small additional vasoconstriction when its concentration was doubled from 10−3 to 2 × 10−3 mol/l. It is concluded that, in the rabbit basilar artery, the vasoconstrictor response to UTP is mediated by a pyrimidine nucleotide receptor which is distinct from the P2-purinoceptor, and that the vasoconstrictor response to ATP is mediated by a P2-receptor which is distinct from the known P2-subtypes.

ATP (1 mM) inhibited, whereas 2-methylthio-ATP (30 μM), a P 2Y -selective purinoceptor agonist, increased electrically evoked release of [ 3 H]noradrenaline from chick sympathetic neurons. The P 2X -selective purinoceptor agonist α,β-methylene-ATP (30 μM) had no effect. The ATP-induced inhibition of release as well as the facilitation caused by 2-methylthio-ATP was not affected by the selective adenosine (P 1 ) receptor antagonist 8-( p -sulfophenyl)-theophylline (8-PST; 100 μM), but completely prevented by the non-selective P 2 antagonist suramin (300 μM). The present data reveal a dual regulation of noradrenaline release from sympathetic neurons. Facilitation seems to be mediated by a P 2Y purinoceptor, whereas inhibition is caused by a P 2 purinoceptor which needs further subtype characterization.

The largest rami caecales of the ileocolic artery (a branch of the mesenteric artery) were perfused at a constant rate of flow. Either vasoconstriction or the release of previously incorporated [3H]noradrenaline was measured. The following opioid agonists inhibited the vasoconstriction elicited by electrical field pulses (5 pulses at 10 Hz; EC50 values in brackets): [Leu5]enkephalin (596 nmol/l), [D-Ala2,D-Leu5]enkephalin (69 nmol/l), dynorphin-(1-13) (366 nmol/l) and ethylketocyclazocine (668 nmol/l). Fentanyl (up to 30 mumol/l) and normorphine (up to 100 mumol/l) caused at best minimal inhibition. The effects of [Leu5]enkephalin and dynorphin-(1-13) were antagonized by naloxone. Only the effect of [Leu5]enkephalin but not that of dynorphin-(1-13) was antagonized by the delta-selective antagonist ICI 154129. [Leu5]Enkephalin and dynorphin-(1-13) did not decrease the vasoconstrictor response to exogenous noradrenaline or ATP. In arteries preincubated with [3H]noradrenaline, electrical stimulation (50 pulses at 1 Hz) increased the outflow of tritium. The stimulation-evoked overflow was reduced by [Leu5]enkephalin and dynorphin-(1-13), and the effect of [Leu5]enkephalin was antagonized by naloxone. It is concluded that the postganglionic sympathetic neurons of the ramus caecalis possess presynaptic opioid receptors which, when activated, inhibited transmitter release. The receptors appear to be of the delta- and kappa- but not the mu-type.

Effects of nucleotides and nucleosides on smooth muscle tension and the release of previously stored [3H]-noradrenaline were studied in the mouse isolated vas deferens. The tissue was stimulated twice by 20 electrical field pulses delivered at 2 Hz (S1, S2). alpha,beta-Methylene-ATP, ATP gamma S, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine (up to 100 mumol/l) and uridine (up to 1 mmol/l). The electrically evoked overflow of tritium was reduced by the drugs in the following order of potency: ATP gamma S greater than ATP = adenosine greater than UTP; alpha,beta-methylene-ATP (up to 10 mumol/l) and uridine (up to 1 mmol/l) did not significantly change the evoked overflow. 8-(p-Sulphophenyl)theophylline did not alter the contractile responses to the nucleotides; it prevented the overflow-inhibiting effect of adenosine and reduced that of UTP; the overflow-inhibiting effects of ATP and ATP gamma S were not significantly attenuated. After prolonged exposure to alpha,beta-methylene-ATP, all contractile nucleotide effects were abolished; in contrast, the depression by adenosine and the nucleotides of the evoked overflow of tritium persisted. None of the effects was changed by indometacin, yohimbine or reactive blue 2. It is concluded that ATP, ATP gamma S, alpha,beta-methylene-ATP and UTP produce contraction of the vas deferens by activation of P2x-receptors. Moreover, the nucleotides inhibit per se the release of [3H]-noradrenaline (and presumably the co-transmitter mixture of noradrenaline and ATP); the effect of ATP is not, or only to a small extent, due to breakdown to adenosine. The presynaptic site of action of the purine nucleotides is a P2-receptor which differs from the P2x-receptor and may be a reactive blue 2-resistant "P2y-like" receptor.

Vasa deferentia from mice were field-stimulated by trains of 10 pulses delivered at 0.5 Hz. The pulses elicited separate twitches, the first of which (corresponding to a single pulse) exceeded the following ones in height and width and often was clearly biphasic. α, β-Methylene-ATP 1 μmol/1 and suramin 100 μmol/1 caused almost identical changes. They reduced the height of the first twitch in the train by about one half and also reduced its width in a manner indicating that only the second, slow phase remained, but reduced much more markedly the following twitches in which now a small second, slow phase also became detectable. Idazoxan 0.1 μmol/1 or yohimbine 0.1 μmol/l, when added in the presence of a, β-methylene-ATP or suramin, further decreased the first twitch but enhanced twitches No. 2 to 10. These responses were then almost abolished by prazosin 0.1 μmol/l. Successive addition of prazosin 0.1 μmol/l and idazoxan 0.1 μmol/1 to previously untreated vasa deferentia depressed the response to the first pulse by about one half in a manner indicating that only the first, rapid phase remained, but had comparatively little effect on the responses to the subsequent pulses. Suramin 100 μmol/l almost abolished the contractions remaining in the presence of prazosin and idazoxan. The results indicate that the first, rapid phase of the neurogenic contractions elicited by single or low frequency pulses is mediated by ATP which substantially contributes to all responses in a train. The second, slow phase is mediated by noradrenaline which substantially contributes to the response to the first pulse only. The adrenergic contribution seems to be mediated by postjunctional α1- as well as a α2-adrenoceptors. Prejunctional α2-adrenergic autoinhibition depresses the release of both ATP and noradrenaline.

Contractions and overflow of tritium and ATP elicited by hypogastric nerve stimulation (HNS) and field stimulation (FS) were studied in the guinea-pig isolated vas deferens preincubated with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. HNS and FS elicited contraction, tritium overflow and ATP overflow. HNS at supramaximal current strength produced smaller responses than did FS at supramaximal current strength (210 pulses/7 Hz). Supramaximal HNS and submaximal FS were used in the remainder of the study. Prazosin (0.3 mumol/l) reduced contractions and the overflow of ATP elicited by both HNS and FS; the evoked overflow of tritium was not changed (210 pulses/7 Hz). Combined administration of prazosin (0.3 mumol/l) and suramin (300 mumol/l) abolished contractions and reduced the overflow of ATP elicited by both HNS and FS slightly more than did prazosin alone; tritium overflow again was not changed (210 pulses/7 Hz). Contractions, tritium overflow and ATP overflow increased with the frequency of both HNS and FS (from 7 to 25 Hz; 210 pulses); the increase in ATP overflow with frequency was more marked than the increase in tritium overflow. The preferential increase of ATP overflow with the frequency of HNS and FS persisted in the combined presence of prazosin (0.3 mumol/l) and suramin (300 mumol/l). The study confirms for HNS, a more physiologic way of sympathetic nerve stimulation, several observations previously obtained with FS. First, HNS-evoked ATP release is detectable as an overflow of ATP into the superfusion fluid.(ABSTRACT TRUNCATED AT 250 WORDS)