adp-beta-s and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5'-[γ-thio]triphosphate (ATPγS) or adenosine 5'-[β-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5'-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPγS- and adenosine 5'-[β-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPγS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions.

Topics: Abscisic Acid; Adenosine Diphosphate; Adenosine Triphosphate; Apyrase; Arabidopsis; Arabidopsis Proteins; Enzyme Inhibitors; Extracellular Space; Gene Expression Regulation, Plant; Hydrogen Peroxide; Light; Models, Biological; Nitric Oxide; Nucleotides; Plant Stomata; Promoter Regions, Genetic; Pyridoxal Phosphate; RNA Interference; Thionucleotides; Triazines

ATP acts as a growth factor as well as a toxic agent by stimulating P2 receptors. The P2 receptor-activated signaling cascades mediating cellular growth and cell survival after injury are only incompletely understood. Therefore, the aim of the present study was to identify the role of the phosphoinositide 3 kinase (PI3-K/Akt) and the mitogen-activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) pathways in P2Y receptor-mediated astrogliosis after traumatic injury and after microinfusion of ADP beta S (P2Y(1,12,13) receptor agonist) into the rat nucleus accumbens (NAc). Mechanical damage and even more the concomitant treatment with ADP beta S, enhanced P2Y(1) receptor-expression in the NAc, which could be reduced by pretreatment with the P2X/Y receptor antagonist PPADS. Quantitative Western blot analysis indicated a significant increase in phosphorylated (p)Akt and pERK1/2 2 h after ADP beta S-microinjection. Pretreatment with PPADS or wortmannin abolished the up-regulation of pAkt by injury alone or ADP beta S-treatment. The ADP beta S-enhanced expression of the early apoptosis marker active caspase 3 was reduced by PPADS and PD98059, but not by wortmannin. Multiple immunofluorescence labeling indicated a time-dependent expression of pAkt and pMAPK on astrocytes and neurons and additionally the colocalization of pAkt, pMAPK, and active caspase 3 with the P2Y(1) receptor especially at astrocytes. In conclusion, the data show for the first time the involvement of PI3-K/Akt-pathway in processes of injury-induced astroglial proliferation and anti-apoptosis via activation of P2Y(1) receptors in vivo, suggesting specific roles of P2 receptors in glial cell pathophysiology in neurodegenerative diseases.

Topics: Adenosine Diphosphate; Androstadienes; Animals; Apoptosis; Astrocytes; Brain Injuries; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gliosis; Male; MAP Kinase Signaling System; Nucleus Accumbens; Phosphatidylinositol 3-Kinases; Phosphorylation; Platelet Aggregation Inhibitors; Proto-Oncogene Proteins c-akt; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Thionucleotides; Up-Regulation; Wortmannin

The neurotransmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10-60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. l-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 microM), by desensitization of P2Y receptors with adenosine 5'-[beta-thio]diphosphate (ADP-betaS) (10 microM), and by the selective P2Y1 receptor blocker 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179) (10 microM). Relaxation and IJPs were also significantly reduced by the K(+) channel blocker apamin (1 microM). Removal of extracellular potassium (K(o)) increased IJP amplitude to 205% of control, whereas return of K(o) 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 microM) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apamin-sensitive K(+) channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Anal Canal; Animals; Apamin; Electric Stimulation; Enteric Nervous System; Enzyme Inhibitors; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Mice; Mice, Inbred BALB C; Motor Neurons; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Neural Inhibition; Neuromuscular Junction; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Potassium; Potassium Channel Blockers; Potassium Channels; Purinergic P2 Receptor Antagonists; Purines; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Synaptic Transmission; Tetrodotoxin; Thionucleotides

This study analysed the contribution of the purinergic system to enteric neurotransmission in the longitudinal muscle of mouse distal colon.. Motor responses to exogenous ATP and to nerve stimulation in vitro were assessed as changes in isometric tension.. ATP induced a concentration-dependent contraction, reduced by 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzene disulphonic acid (PPADS), suramin, P2Y purinoreceptor desensitisation with adenosine 5'-O-2-thiodiphosphate (ADPbetaS), and atropine, but unaffected by P2X purinoceptor desensitisation with alpha,beta-methylene ATP (alpha,beta-meATP) and by 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester (MRS 2395), a P2Y(12) selective antagonist. The response to ATP was increased by 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS 2179), a P2Y(1) selective antagonist, tetrodotoxin (TTX) or N(omega)-nitro-L-arginine methyl ester (L-NAME). ADPbetaS, a P2Y-purinergic agonist, induced muscular contraction, with the same pharmacological profile as the ATP-induced contraction. ADP, a natural ligand for P2Y(1) receptors, induced muscular relaxation, antagonized by MRS 2179 and by TTX or L-NAME. Nerve stimulation elicited a transient nitrergic relaxation, followed by contraction. Contractile responses was reduced by atropine, PPADS, suramin, P2Y purinoceptor desensitisation, but not by P2X purinoceptor desensitisation, MRS 2179 or MRS 2395. None of the purinergic antagonists modified the nerve-evoked relaxation.. In the longitudinal muscle of mouse distal colon, ATP, through ADPbetaS-sensitive P2Y purinoceptors, contributed to the excitatory neurotransmission acting directly on smooth muscle and indirectly via activation of cholinergic neurons. Moreover, P2Y1 purinoceptors appear to be located on nitrergic inhibitory neurons. This study provides new insights into the role of purines in the mechanism inducing intestinal transit in mouse colon.

Topics: Adenine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Colon; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Neurotransmitter Agents; Purinergic Agonists; Pyridoxal Phosphate; Thionucleotides; Valerates

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2-methylthioATP (2-MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) increased the food intake after intracerebroventricular infusion in 18-h food-deprived rats. This effect was abolished by pretreatment with the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) or the selective P2Y1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPbetaS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methylester (L-NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co-localization of P2Y1 receptor- and nNOS-immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L-NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Y1-receptor-mediated nitric oxide production.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Behavior, Animal; Dose-Response Relationship, Drug; Drug Interactions; Eating; Enzyme Inhibitors; Fluorescent Antibody Technique; Food Deprivation; Glial Fibrillary Acidic Protein; Hypothalamus; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type I; Nucleotides; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Signal Transduction; Thionucleotides

In the 1st part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex (PFC) were evoked by electrical stimulation of layer I. Recordings with intracellular sharp, microelectrodes showed a concentration-dependent inhibition of the EPSP by adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), when given alone depressed the EPSP and in addition antagonized the effect of ADP-beta-S. Exclusion of the N-methyl-D-aspartate (NMDA) component of the EPSP by D(.)-amino-5-phosphonopentanoic acid (AP-5) abolished the ADP-beta-S-induced depression. The pressure-application of both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused reproducible depolarizations. ADP-beta-S inhibited the effect of NMDA, but did not alter that of AMPA. PPADS was also under these conditions antagonistic with ADP-beta-S. In the 2nd part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ADP-beta-S caused a concentration-dependent inhibition of the responses to NMDA. PPADS alone did not alter the NMDA-currents but again antagonized the action of ADP-beta-S; 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) also abolished the NMDA effect. The ADP-beta-S-induced inhibition persisted in the presence of tetrodotoxin (TTX) or guanosine 5'-O-(3-thiodiphosphate) (GDP-beta-S) applied to the external medium and the pipette solution, respectively. The 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) moderately decreased the ADP-beta-S effect. The inhibitory function of ADP-beta-S on EPSPs and the interaction with PPADS was observed also in layer V pyramidal neurons of the parietal somatosensory cortex. In conclusion, metabotropic P2Y(1) receptors appear to exert a new modulatory influence on fast excitatory amino acid transmission in the cerebral cortex.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Algorithms; Animals; Cerebral Cortex; Electric Stimulation; Excitatory Postsynaptic Potentials; Male; Membrane Potentials; Microelectrodes; Parietal Lobe; Patch-Clamp Techniques; Prefrontal Cortex; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyramidal Cells; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic P2Y1; Reflex, Monosynaptic; Thionucleotides

P2 receptors are important regulators of cerebrovascular tone. However, there is functional heterogeneity of P2Y receptors along the vascular tree, and the functionality of P2Y receptors in small arterioles has not been studied in detail. We investigated the effects of activating P2Y1 and P2Y2 receptors and their underlying dilator mechanisms in rat intracerebral arterioles.. We used computer-aided videomicroscopy to measure diameter responses from isolated and pressurized rat penetrating arterioles (39.9+/-1.2 microm) to the natural P2 receptor agonist ATP in addition to ADP-beta-S (P2Y1-selective) and ATP-gamma-S (P2Y2-selective) and inhibitors of signaling pathways.. Extraluminal application of ATP-gamma-S and ADP-beta-S initiated a biphasic response (initial constriction followed by the secondary dilation) similar to ATP-induced responses. Pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (0.1 mmol/L; a P2Y1 receptor antagonist) blocked ADP-beta-S- but not ATP-gamma-S-induced dilation and affected the ATP-mediated dilation at low concentrations. Nomega-Monomethyl-l-arginine partially inhibited the dilation of ATP and ADP-beta-S but not ATP-gamma-S. High K+ saline suppressed the dilation of all agonists. Indomethacin had no effect.. Both P2Y1 and P2Y2 receptors are functionally present in cerebral arterioles. ATP stimulates P2Y1 receptors at low concentrations, while high concentrations of ATP activate P2Y2 in addition to P2Y1 receptors. Nitric oxide is involved in P2Y1 but not P2Y2 receptor activation. Potassium channels play an important role in the regulation of P2Y receptor-mediated dilation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Arterioles; Brain; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrogen-Ion Concentration; In Vitro Techniques; Indomethacin; Male; Microscopy, Video; Nitric Oxide; omega-N-Methylarginine; Potassium Chloride; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y2; Thionucleotides; Vascular Patency; Vasodilation

Purinergic nucleotides, including ATP and adenosine, are important neuromodulators of peripheral auditory and visual sensory systems (Thorne and Housley, 1996). ATP released by the olfactory epithelium (OE) after noxious stimuli provides a physiological source for a neuromodulatory substance independent of efferent innervation. Here we show that multiple subtypes of purinergic receptors are differentially expressed in olfactory receptor neurons and sustentacular support cells. Activation of purinergic receptors evoked inward currents and increases in intracellular calcium in cultured mouse olfactory receptor neurons. A mouse olfactory epithelial slice preparation and confocal imaging were used to measure changes in intracellular calcium in response to odors, purinergic receptor (P2R) agonists, or combined odor + P2R agonists. Pharmacological studies show that both P2Y and P2X receptor activation by exogenous and endogenous ATP significantly reduces odor responsiveness. Moreover, purinergic receptor antagonists increase the odor-evoked calcium transient, providing direct evidence that endogenous ATP modulates odor sensitivity via activation of multiple purinergic receptor subtypes in olfactory receptor neurons. Odor activation of G-protein-coupled receptors results in increased cAMP production, opening of cyclic nucleotide-gated channels, influx of Ca2+ and Na+, depolarization of the membrane, and activation of voltage- and Ca2+-gated ion channels. On-cell current-clamp recordings of olfactory receptor neurons from neonatal mouse slices revealed that ATP reduced cyclic nucleotide-induced electrical responses. These data also support the idea that ATP modulates odor sensitivity in mammalian olfactory neurons. Peripheral ATP-mediated odor suppression is a novel mechanism for reduced olfactory sensitivity during exposure to olfactotoxins and may be a novel neuroprotective mechanism.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Cells, Cultured; Cyclic AMP; Cyclic GMP; In Vitro Techniques; Male; Membrane Potentials; Mice; Olfactory Mucosa; Olfactory Receptor Neurons; Patch-Clamp Techniques; Pyridoxal Phosphate; Rats; Rats, Inbred Strains; Receptors, Purinergic; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2Y2; RNA, Messenger; Sensory Thresholds; Smell; Stimulation, Chemical; Thionucleotides

Intracellular recordings were made in a mid-pontine slice preparation of the rat brain containing the nucleus locus coeruleus. Focal electrical stimulation evoked biphasic synaptic potentials consisting of early depolarizing (d.p.s.p.) and late hyperpolarizing (i.p.s.p.) components. The alpha(2)-adrenoceptor antagonist idazoxan inhibited the i.p.s.p. without altering the d.p.s.p. All of the following experiments were carried out in the presence of kynurenic acid and picrotoxin to block the glutamatergic and GABAergic fractions of the d.p.s.p., respectively. Guanethidine, which is known to inhibit noradrenaline and ATP release from nerve terminals of postganglionic sympathetic nerves, depressed both the d.p.s.p. and the i.p.s.p. in a concentration-dependent manner. Damage of catecholaminergic nerve terminals by 6-hydroxydopamine also decreased both the d.p.s.p. and the i.p.s.p. The P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) depressed the d.p.s.p., whereas the i.p.s.p. remained unaffected. The further application of PPADS did not increase the depression of the d.p.s.p. by guanethidine. Superfusion with the mixed alpha-adrenoceptor agonist noradrenaline or the selective P2 receptor agonist adenosine 5'-O-(2-thiodiphosphate) inhibited both the d.p.s.p. and the i.p.s.p. The inhibitory effects of these agonists were prevented by the respective antagonists idazoxan or suramin. In the presence of suramin noradrenaline failed to inhibit the residual d.p.s.p. Superfused noradrenaline potentiated rather than inhibited responses to pressure-applied alpha,beta-methylene-ATP; superfused adenosine 5'-O-(2-thiodiphosphate) did not interact with pressure-applied noradrenaline. In conclusion, we present electrophysiological evidence for the co-release of ATP and catecholamines in the CNS. At the cell somata of neurons in the locus coeruleus, noradrenaline and ATP activate inhibitory alpha(2)-adrenoceptors and excitatory P2 receptors, respectively. In addition, inhibitory presynaptic autoreceptors of the alpha(2) and P2 types appear to regulate release of the two co-transmitters.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Catecholamines; Electric Stimulation; Evoked Potentials; Excitatory Postsynaptic Potentials; Guanethidine; Idazoxan; In Vitro Techniques; Kynurenic Acid; Male; Membrane Potentials; Neurons; Norepinephrine; Oxidopamine; Picrotoxin; Pons; Pyridoxal Phosphate; Rats; Rats, Wistar; Synaptic Transmission; Thionucleotides

The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5'-O-2-thiodiphosphate, (ADPbetaS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N'-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y(1) receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N'-acetyl-5-HT production is a P2Y(1)-like receptor, coupled to a G protein which stimulates phospholipase C.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adrenergic alpha-Agonists; Animals; Cells, Cultured; Cyclic AMP; Female; Male; Norepinephrine; Phosphodiesterase Inhibitors; Pineal Gland; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Serotonin; Signal Transduction; Thionucleotides; Type C Phospholipases

The possibility was tested that endogenous ATP released upon alpha(1)-adrenoceptor activation causes relaxation of the rat vas deferens smooth muscle. ATP, 2-methylthio ATP and adenosine relaxed the vas deferens precontracted with 80 mM K(+). The metabolically stable P2 receptor agonists alpha,beta-methylene ATP (alpha,beta-MeATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) had little or no effect. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline did not significantly affect the response to ATP. The P2 receptor antagonist reactive blue 2 markedly reduced the relaxation (by up to 73%); suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and acid blue 129 caused no change. ATP, but not alpha,beta-MeATP, also attenuated contractions elicited by noradrenaline at resting tension; reactive blue 2 blocked the inhibitory effect of ATP. Reactive blue 2, by itself, enhanced the response to noradrenaline (by up to 36%); suramin, PPADS and acid blue 129 caused no change. In the presence of the ATP-degrading enzymes apyrase and nucleotide pyrophosphatase, the facilitatory effect of reactive blue 2 was lost. Apyrase, by itself, enhanced the response to noradrenaline (by 13%). The results indicate that endogenous ATP, released from rat vas deferens smooth muscle upon alpha(1)-adrenoceptor stimulation, causes relaxation. The site of action of ATP is not a typical smooth muscle P2Y receptor.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Apyrase; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Muscle Relaxation; Norepinephrine; Nucleotides; Potassium; Purinergic P1 Receptor Antagonists; Pyridoxal Phosphate; Pyrophosphatases; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Suramin; Theophylline; Thionucleotides; Triazines; Vas Deferens; Vasoconstrictor Agents

1. In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (alpha,beta-methylene adenosine 5'-triphosphate; alpha,beta-meATP), P2Y1,12 (adenosine 5'-O-(2-thiodiphosphate; ADP-beta-S) and P2Y2,4,6 receptors (uridine 5'-O-(3-thiotriphosphate; UTP-gamma-S). Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) was used as a non-selective antagonist. The astroglial reaction was studied by means of immunocytochemical double-labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5-bromo-2'-deoxyuridine (BrdU). 2. The agonist-induced changes in comparison to the artificial cerebrospinal fluid (aCSF)-treated control side reveal a strong mitogenic potency of ADP-beta-S and alpha,beta-meATP, whereas UTP-gamma-S was ineffective. The P2 receptor antagonist PPADS decreased the injury-induced proliferation when given alone and in addition inhibited all agonist effects. 3. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up-regulation of GFAP. A significant increase of both GFAP-immunoreactivity (IR) and GFA-protein content (by using Western blotting) was found after microinfusion of alpha,beta-meATP or ADP-beta-S. In contrast, UTP-gamma-S failed to increase the GFAP-IR. The morphogenic effects were also inhibited by pre-treatment with PPADS. 4. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP-labelled astrocytes. 5. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Astrocytes; Glial Fibrillary Acidic Protein; Gliosis; Immunohistochemistry; Male; Neuroglia; Nucleus Accumbens; Pyridoxal Phosphate; Rats; Rats, Wistar; Thionucleotides; Uridine Triphosphate

Purinoceptor (P2X and P2Y) mediated Ca2+ signaling in cultured human microglia was studied using Ca2+ sensitive fluorescence microscopy. ATP (at 100 microM) induced a transient increase in [Ca2+]i in both normal and Ca(2+)-free solution suggesting a primary contribution by release from intracellular stores. This conclusion was further supported by the failure of ATP to cause a divalent cationic influx in Mn2+ quenching experiments. However, when fluorescence quenching was repeated after removal of extracellular Na+, ATP induced a large influx of Mn2+, indicating that inward Na+ current through a non-selective P2X-coupled channel may normally suppress divalent cation influx. Inhibition of Mn2+ entry was also found when microglia were depolarized using elevated external K+ in Na(+)-free solutions. The possibility of P2X inhibition of Ca2+ influx was then investigated by minimizing P2X contributions of purinergic responses using either the specific P2Y agonist, ADP-beta-S in the absence of ATP or using ATP combined with PPADS, a specific inhibitor of P2X receptors. In quenching studies both procedures resulted in large increases in Mn2+ influx in contrast to the lack of effect observed with ATP. In addition, perfusion of either ATP plus PPADS or ADP-beta-S alone caused a significantly enhanced duration (about 200%) of the [Ca2+]i response relative to that induced by ATP. These results show that depolarization induced by P2X-mediated Na+ influx inhibits store-operated Ca2+ entry resulting from P2Y activation, thereby modulating purinergic signaling in human microglia.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Calcium; Calcium Signaling; Culture Media; Fluorescence; Humans; Ion Channels; Manganese; Microglia; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Thionucleotides

1 Adenosine 5'-triphosphate (ATP) is an enteric neurotransmitter which acts at purine receptors on intestinal nerve and muscle. This study set out to shed light on the receptor mechanisms by which exogenous and endogenous ATP influences intestinal peristalsis. 2 Peristalsis in isolated segments of the guinea-pig small intestine was triggered by a perfusion-induced rise of the intraluminal pressure. Motor changes were quantified by alterations of the peristaltic pressure threshold (PPT) at which propulsive muscle contractions were elicited. 3 ATP (>/= 3 microM) increased PPT and abolished peristalsis at concentrations of 100-300 microM. Adenosine 5'-O-2-thiodiphosphate (ADPbetaS, 3-100 microM) was more potent, whereas alpha,beta-methylene ATP (alpha,beta-meATP, 3-100 microM) was less potent, than ATP in depressing peristalsis. 4 8-Phenyltheophylline (10 microM) attenuated the anti-peristaltic effect of 10 and 30 microM ATP but not that of higher ATP concentrations. Apamin (0.5 microM) counteracted the ability of ATP, ADPbetaS and alpha,beta-meATP to enhance PPT. Suramin (300 microM) and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 150 microM) antagonized the inhibitory effect of alpha,beta-meATP on peristalsis but did not alter the effect of ATP and ADPbetaS. 5 PPADS (50-150 microM) reduced PPT by as much as 50%. This stimulant effect on peristalsis was prevented by suramin (300 microM) but left unaltered by apamin (0.5 microM) and NG-nitro-L-arginine methyl ester (300 microM). 6 These data show that exogenous and endogenous ATP inhibits intestinal peristalsis via different apamin-sensitive purinoceptor mechanisms. Exogenous ATP depresses peristalsis mostly via suramin- and PPADS-insensitive P2 receptors, whereas endogenous purines act via P2 receptors sensitive to both suramin and PPADS.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Apamin; Female; Guinea Pigs; Ileum; In Vitro Techniques; Intestines; Male; Peristalsis; Purines; Pyridoxal Phosphate; Receptors, Purinergic; Receptors, Purinergic P2; Suramin; Thionucleotides

1. By using the sucrose gap technique, we have investigated the effect of the metabolically stable P2Y receptor agonist, adenosine 5'-O-2-thiodiphosphate (ADPbetaS), on the membrane potential and tension in the circular muscle of the guinea-pig proximal colon. All experiments were performed in the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.1 microM) and GR 94800 (0.1 microM), respectively. 2. ADPbetaS (100 microM for 15 s) evoked a tetrodotoxin- (1 microM) resistant hyperpolarization and contraction of the smooth muscle. In the presence of apamin (0.1 microM), the ADPbetaS-induced hyperpolarization was converted to depolarization and the contraction was potentiated while tetraethylammonium (TEA, 10 mM) did not affect significantly the response to ADPbetaS. The combined application of apamin and TEA reproduced the effect observed with apamin alone. 3. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acids (PPADS, 30 microM) slightly but significantly increased the ADPbetaS-induced hyperpolarization, while the contraction evoked by ADPbetaS was reduced by about 80%. Suramin (100 microM) did not affect the ADPbetaS-induced hyperpolarization but totally blocked the ADPbetaS-induced contraction. In the presence of suramin (100 microM), a small relaxation of the circular muscle was observed upon application of ADPbetaS. 4. The contraction and hyperpolarization evoked by ADPbetaS were abolished in Ca2+-free Krebs solution. The blocker of sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (10 microM) reduced contraction and hyperpolarization induced by ADPbetaS by about 60 and 50%, respectively. 5. A comparison of our present and previous findings enables to conclude that at least 3 types of P2 receptors are present on the smooth muscle of the guinea-pig colon, as follows: (1) inhibitory P2 receptors, producing an apamin-sensitive hyperpolarization, which are activated by alpha,beta-methylene ATP (alpha,beta-meATP) and by endogenously released purines, sensitive to suramin and PPADS; (2) inhibitory P2 receptors, producing an apamin-sensitive hyperpolarization, which are activated by ADPbetaS and are resistant to suramin and PPADS; (3) excitatory P2 receptors, producing contraction, which are activated by ADPbetaS and are sensitive to suramin and PPADS. The data also support the idea of the exis

Topics: Adenosine Diphosphate; Animals; Apamin; Autonomic Nervous System; Colon; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Indoles; Male; Muscle Contraction; Muscle, Smooth; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Agonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Suramin; Thionucleotides; Vasodilator Agents

Effects of purinoceptor antagonists on the relaxant responses to adenine nucleotides were examined to characterize the subtypes of P2-receptor in rat gastric circular muscle. In tissues contracted by acetylcholine, a P2-receptor antagonist, suramin (100 microM), inhibited the relaxant responses to ATP, adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) and alpha,beta-methylene ATP but not that to adenosine, while a P1-receptor antagonist, 8-phenyltheophylline (3 microM) did vice versa. The inhibitory effect of suramin was more potent for the relaxant responses to alpha,beta-methylene ATP than those to ATP or ADPbetaS. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (3-30 microM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) (30 and 100 microM) inhibited the relaxation caused by alpha,beta-methylene ATP but not by ATP, ADPbetaS or adenosine. These results suggest that ATP and ADPbetaS cause relaxation via the classical P2Y receptors resistant to PPADS and DIDS. In addition, alpha,beta-methylene ATP causes relaxation via the distinct P2 receptors sensitive to PPADS and DIDS in rat gastric circular muscle.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antineoplastic Agents; Apamin; Dose-Response Relationship, Drug; Gastric Mucosa; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Stomach; Suramin; Thionucleotides

Extracellular recording techniques were used in brain slices to characterize excitatory responses produced by purine nucleotides in the rat medial vestibular nucleus, an area where functional purinoceptors have not previously been described. In the continued presence of the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine, which alone caused a small increase in the spontaneous firing rate, the P2 purinoceptor agonists alpha,beta-methyleneadenosine 5'-triphosphate (alphabeta meATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) caused concentration-dependent increases in spontaneous firing rate, with EC50 values of 41.8 and 1.7 microM, respectively. Only approximately 35% of all neurons studied displayed excitatory responses to these agents. Responses waned in the continued presence of high concentrations of the latter, but not the former agonist. Furthermore, in the continued presence of a maximal concentration of alphabeta meATP, ADPbetaS produced further increases in the firing rate of these neurons. The P2 antagonist, suramin, ablated responses to alphabeta meATP, but did not affect responses to ADPbetaS, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid antagonized responses to both agonists. The nucleotide analogue alpha,beta-methyleneadenosine 5'-diphosphate, which displays affinity for putative P2X receptors in brain, also produced concentration-dependent increases in firing frequency, which were also markedly antagonized in the presence of suramin, this agonist being only slightly less potent than alphabeta meATP. In conclusion, a subpopulation of rat medial vestibular neuronal responses mediated by both P2X and P2Y purinoceptors can be distinguished. Comparison of their properties with those of recombinantly expressed P2X and P2Y receptors suggests that these endogenous P2 purinoceptor types differ in several important aspects from heterologously expressed recombinant receptors identified from cloning studies.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Evoked Potentials; In Vitro Techniques; Male; Neurons; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Theophylline; Thionucleotides; Time Factors; Vestibular Nuclei