2-methylthioadenosine Triphosphate Research Articles

ATP-Induced Currents Carried Through P2X7 Receptor in Rat Myometrial Cells

The functional significance of purinergic P2 receptors in the myometrium is unclear. We previously reported the ATP-induced ionic currents in rat myometrial cells, causing uterine contractility. The aim of this study is to determine the main P2X receptors that carry the adenosine triphosphate (ATP)-induced currents. We cloned predominantly expressed P2X7 receptors from rat myometrium and transfected into cultured COS-7 cells. Reconstructed P2X7 receptor currents were characterized using the whole-cell patch clamp method. Extracellular ATP induced currents through P2X7 receptors with effective concentration (EC(50)) of 155 μmol/L, without desensitization. The myometrial P2X7 receptor was permeable to multiple monovalent cations with conductances ranked as K(+)>Cs(+)>Li(+)>Na(+). It was activated by P2X receptor agonists, with effectiveness ranked as 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP)>>ATP>αβ-methylene ATP (αβ-MeATP)>2-methylthio ATP (2-MeSATP)>uridine triphosphate (UTP)>guanosine triphosphate (GTP)>adenosine diphosphate (ADP). These currents were blocked by selective P2X7 receptor antagonists and extracellular Mg(2+). P2X7 receptors carry ATP-induced currents in rat myometrial cells.

Higher Concentrations of Extracellular ATP Suppress Proliferation of Caco-2 Human Colonic Cancer Cells via an Unknown Receptor Involving PKC Inhibition

Background/Aims: Adenosine 5′-triphosphate (ATP) mediates a variety of signal transductions via ATP receptors such as P2X and P2Y receptors. The present study aimed at understanding the mechanism underlying extracellular ATP-induced suppression of Caco-2 human colonic cancer cell proliferation. Methods: Caco-2 cells were cultured. To examine cell viability and cell cycling, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, fluorescent cytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and flow cytometry were carried out. To see mRNA expression of ATP receptors, reverse transcription-polymerase chain reaction (RT-PCR) was performed. To examine PKC activity and mitogen-activated protein (MAP) kinase activity, in situ PKC assay and Western blotting using an anti-extracellular signal-regulated kinase 1 (ERK1)-antibody and an anti-phospho-ERK antibody were carried out. Results: Extracellular ATP or the unhydrolyzed ATP analogue 5′-adenylyimido-diphosphate (AMP-PNP) reduced Caco-2 cell viability in a concentration (10 µM-10 mM)-dependent manner at 48-h treatment, and the effect was not affected by caspase inhibitors. Caco-2 cells were little reactive to propidium iodide and Hoechst 33342 or little positive to TUNEL after 48-h treatment with ATP (1 mM). In the flow cytometry, 48-h treatment with ATP (1 mM) arrested cell cycling at the S phase in Caco-2 cells. P₂ purinoceptor agonists reduced Caco-2 cell viability with the order of potency: 2-methylthio ATP>UTP>β, γ-methylene ATP, and the ATP effect was partially inhibited by suramin, a non-selective inhibitor of P₂ purinoceptors. The PKC inhibitor GF109203X or the MAP kinase kinase inhibitor PD98059 reduced Caco-2 cell viability to an extent similar to that achieved by ATP (1 mM), and no further reduction was obtained with co-treatment with ATP. ATP and its ATP analogues such as AMP-PNP and ATPγS, at higher concentrations (1-10 mM), inhibited PKC activation in Caco-2 cells in a fashion that mimics the effect of GF109203X, but PD98059 exhibited no effect on PKC activation. The inhibitory effect of ATP on PKC activation was not found with SK-N-SH cells, a human neuroblastoma cell line, but the cells expressed all the mRNAs for P2X and P2Y receptors that Caco-2 cells did. ATP (10 mM) or GF109203X inhibited activation of ERK, a MAP kinase, in Caco-2 cells. Conclusion: Extracellular ATP, at higher concentrations, suppresses Caco-2 cell proliferation at the S phase of cell cycling by inhibiting PKC, possibly as mediated via an unknown ATP receptor, followed by MAP kinase.

Osmosensitive response of glutamate in the substantia nigra

Previous studies have suggested the increase of extracellular glutamate (GLU) in the substantia nigra (SN) as a cause of dopamine-cell degeneration (excitotoxicity) in Parkinson's disease (PD). However, the mechanisms involved in this increase remain unknown. The present work studied osmoregulation as a cause of GLU release in the SN. Microdialysis was used to change extracellular osmolarity, to administer drugs and to quantify the extracellular non-synaptic GLU (EnS-GLU). Two osmolarity modifications were performed, a moderate decrease (5%) resembling physiological modifications and a substantial decrease (≥ 20% decrease) similar to that observed under pathological conditions. Hypo-osmolarity induced a dose–response (285–80 mOsm) increase of EnS-GLU which was detected after small osmolarity modifications (15 mOsm) and which was very marked (> 1000%) after more intense osmolarity changes. This response disappeared after pre-treating rats with a P2 purinergic-receptor antagonist (pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid; 1 mM) suggesting ATP involvement in the osmosensitive EnS-GLU response. The EnS-GLU increase observed after administration of ATP (0.1–100 μM) and 2-methylthioadenosine triphosphate tetrasodium (P2-receptor agonist; 100 μM) and the lack of effects of adenosine administration (1 mM) suggest that the ATP action on P2 receptors is an amplificatory mechanism in the osmosensitive EnS-GLU response. The marked action of osmolarity on extracellular Glu suggests osmolarity regulation as a possible source for excitotoxicity in the SN.

Pharmacological properties of P2 receptors on rat otic parasympathetic ganglion neurons

To elucidate the pharmacological profile of P2X receptors and the probable expression of P2Y receptors in otic ganglion neurons from 17-day-old rats, single neurons were enzymatically isolated and maintained in tissue culture for up to 30 h. Whole-cell voltage-clamp recording was carried out at a holding potential of − 60 mV. Most otic ganglion neurons responded to adenosine 5′-triphosphate (ATP), 2-methylthio ATP (2-MeSATP) and α,β-methylene ATP (αβ-meATP) with sustained currents and EC 50 values of 19 µM, 47 µM and 94 µM, respectively. 2′,3′- O-trinitrophenyl-ATP (TNP-ATP) inhibited the response to αβ-meATP and ATP with an IC 50 values of 3.9 nM and 18.3 nM, respectively, which was closed to that observed in nodose neurons. The response to ATP was antagonized by suramin and cibacron blue. The dose–response curve of suramin against ATP response at a pH of 6.5 was shifted to the left compared to that at a pH of 7.4. Diinosine pentaphosphate (Ip 5I), which blocks P2X 3, but not P2X 2/3-mediated responses, had no effect on the currents evoked by ATP or αβ-meATP. In some neurons, uridine 5′-triphosphate (UTP) induced a tiny, but long-lasting current with a mean amplitude of 0.034 ± 0.011 nA. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the expression of mRNAs for P2X 2, P2X 3, P2X 4, P2X 6 and P2X 7, but not for P2X 1 and P2X 5 receptors in otic ganglion. In conclusion, in rat otic ganglion neurons, P2X 2/3 heteromultimer receptors dominate, but P2X 7 and P2Y 2 or P2Y 4 receptors also play roles.

Distinct P2Y Receptor Subtypes Regulate Calcium Signaling in Human Retinal Pigment Epithelial Cells

Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells. Intracellular Ca(2+) levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis. Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca(2+)](i) in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca(2+) influx component. ATP and UTP were equipotent (EC(50), 6 muM) and maximum responses were equivalent, suggesting activation of a P2Y(2) receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC(50)s of 1 muM and 0.3 muM. The P2Y(1) antagonist MRS 2179 (10 muM) inhibited these responses, confirming functional expression of P2Y(1) receptors. The presence of a response to UDP suggested P2Y(6) expression. There was no influx component to P2Y(1)- and P2Y(6)-mediated responses. mRNA for P2Y(1), P2Y(2,) P2Y(4), and P2Y(6) receptor subtypes was found in cultured RPE cells, and for P2Y(1), P2Y(2,) P2Y(4,) P2Y(6), and P2Y(12) it was found in native RPE cells. Expression of P2Y(1), P2Y(2), and P2Y(6) protein was found in native and cultured RPE cells. These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.

Endothelium-dependent vasodilation of cerebral arteries is altered with simulated microgravity through nitric oxide synthase and EDHF mechanisms

Cephalic elevations in arterial pressure associated with microgravity and prolonged bed rest alter cerebrovascular autoregulation in humans. Using the head-down tail-suspended (HDT) rat to chronically induce headward fluid shifts and elevate cerebral artery pressure, previous work has likewise shown cerebral perfusion to be diminished. The purpose of this study was to test the hypothesis that 2 wk of HDT reduces cerebral artery vasodilation. To test this hypothesis, dose-response relations for endothelium-dependent (2-methylthioadenosine triphosphate and bradykinin) and endothelium-independent (nitroprusside) vasodilation were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (10(-5) M) and cyclooxygenase inhibitor indomethacin (10(-5) M). MCA caveolin-1 protein content was measured by immunoblot analysis. Endothelium-dependent vasodilation to 2-methylthioadenosine triphosphate and bradykinin were both lower in MCAs from HDT rats. These lower vasodilator responses were abolished with N(G)-nitro-L-arginine methyl ester but were unaffected by indomethacin. In addition, HDT was associated with lower levels of MCA caveolin-1 protein. Endothelium-independent vasodilation was not altered by HDT. These results indicate that chronic cephalic fluid shifts diminish endothelium-dependent vasodilation through alterations in the endothelial nitric oxide synthase signaling mechanism. Such decrements in endothelium-dependent vasodilation of cerebral arteries could contribute to the elevations in cerebral vascular resistance and reductions in cerebral perfusion that occur after conditions of simulated microgravity in HDT rats.

Experimental and Modeling Studies of Desensitization of P2X3 Receptors

The function of ATP-activated P2X3 receptors involved in pain sensation is modulated by desensitization, a phenomenon poorly understood. The present study used patch-clamp recording from cultured rat or mouse sensory neurons and kinetic modeling to clarify the properties of P2X3 receptor desensitization. Two types of desensitization were observed, a fast process (t1/2 = 50 ms; 10 microM ATP) following the inward current evoked by micromolar agonist concentrations, and a slow process (t1/2 = 35 s; 10 nM ATP) that inhibited receptors without activating them. We termed the latter high-affinity desensitization (HAD). Recovery from fast desensitization or HAD was slow and agonist-dependent. When comparing several agonists, there was analogous ranking order for agonist potency, rate of desensitization and HAD effectiveness, with 2-methylthioadenosine triphosphate the strongest and beta,gamma-methylene-ATP the weakest. HAD was less developed with recombinant (ATP IC50 = 390 nM) than native P2X3 receptors (IC50 = 2.3 nM). HAD could also be induced by nanomolar ATP when receptors seemed to be nondesensitized, indicating that resting receptors could express high-affinity binding sites. Desensitization properties were well accounted for by a cyclic model in which receptors could be desensitized from either open or closed states. Recovery was assumed to be a multistate process with distinct kinetics dependent on the agonist-dependent dissociation rate from desensitized receptors. Thus, the combination of agonist-specific mechanisms such as desensitization onset, HAD, and resensitization could shape responsiveness of sensory neurons to P2X3 receptor agonists. By using subthreshold concentrations of an HAD-potent agonist, it might be possible to generate sustained inhibition of P2X3 receptors for controlling chronic pain.

Adenosine 5′-triphosphate and noradrenaline are excitatory cotransmitters to the fibromuscular stroma of the guinea pig prostate gland

Immunohistochemical studies demonstrated abundant P2X 1-receptor immunoreactivity colocalized with α-actin within the fibromuscular stroma of the guinea pig prostate. P2X 2-, P2X 3- and P2X 4-receptor immunoreactivity was absent. αβmethylene Adenosine 5′-triphosphate (ATP) attenuated contractile responses to electrical field stimulation (50 V, 0.5 ms, 5–20 Hz) in the absence and presence of prazosin (0.3 μM). Responses to 1–2 Hz were unaffected. ARL 67156 (6- N, N-Diethyl-β-γ-dibromomethylene- d-adenosine-5-triphosphate; 100 μM) enhanced contractile responses to electrical field stimulation (50 V, 0.5 ms, 10–20 Hz). Concentration–response curves to exogenously applied ATP analogues on unstimulated preparations elicited concentration-dependent suramin (100 μM)-sensitive contractions. The rank order of potency was: αβmethylene ATP>2methylthio ATP=βγmethylene ATP>adenosine 5′-diphosphate (ADP)=ATP. Adenosine and adenosine 5′-monophosphate (AMP) did not produce contractile responses. These results demonstrate the presence of functional P2X 1-receptors within the fibromuscular stroma of the guinea pig prostate and suggest a cotransmitter role for ATP with noradrenaline during high-frequency stimulation.

P2 receptors in the murine gastrointestinal tract

The actions of adenosine, adenosine 5′-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), α,β-methylene ATP (α,β-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y 1, P2Y 2, P2Y 4, P2X 1 and P2X 2 receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y 1 mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP≥2-MeSATP>α,β-meATP>ATP=UTP=adenosine. This is suggestive of P2Y 1-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to α,β-meATP. As ATP and UTP are equipotent, the presence of a P2Y 2 receptor is indicated. ATP responses were inhibited by the P2Y 1-selective antagonist MRS 2179, and suramin. P2Y 1 receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y 1 mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and N ω-nitro- l-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>α,βmeATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X 2 receptors. Immunoreactivity to P2X 2 receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y 1 receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y 1, P2Y 2 and a novel P2Y receptor subtype responsive to α,β-meATP in smooth muscle, while P2X 2 receptors mediate contraction of colonic smooth muscle.

Extracellular ATP Is an Autocrine/Paracrine Regulator of Hypoxia-induced Adventitial Fibroblast Growth: SIGNALING THROUGH EXTRACELLULAR SIGNAL-REGULATED KINASE-1/2 AND THE Egr-1 TRANSCRIPTION FACTOR

Important autocrine/paracrine functions for the adenine nucleotides have been proposed in several tissues. We addressed the possibility that extracellular ATP would modulate/mediate hypoxia-induced adventitial fibroblast growth. Acute hypoxia (3% O(2), 10-60 min) increased extracellular ATP concentrations in adventitial fibroblasts and in lung microvascular endothelial cells, and chronic hypoxia (3% O(2), 14-30 days) markedly attenuated the rate of extracellular ATP hydrolysis by ecto-nucleotidase(s). Exogenous ATP stimulated [(3)H]thymidine incorporation in fibroblasts as did UTP, ADPbeta, 2-methylthioadenosine triphosphate, adenosine 5'-(alpha,beta-methylene)triphosphate, and benzoylbenzoyl-ATP (2'-3'-O-(4-benzoylbenzoyl)-ATP), indicating that both P2Y and P2X purinoceptors can mediate mitogenic responses. Suramin (100 microm), Cibacron blue 3GA (100 microm), and pyridoxalphosphate-6-azophenyl-2',-4'-disulfonic acid (100 microm) as well as apyrase (5 units/ml) attenuated hypoxia- and ATP-induced and DNA synthesis, indicating activation and a functional role of purinoceptors under hypoxic conditions. ATP-induced DNA synthesis was augmented by hypoxia in an additive fashion, whereas ATP and hypoxia synergistically increased growth factor-induced DNA synthesis, again suggesting that ATP and hypoxia utilize similar signaling pathways to induce proliferation. Indeed, we found that ATP (100 microm) and hypoxia (3% O(2)) induced expression and activation of Egr-1 transcription factor, and both stimuli acted, in part, through a G(alpha)(i)/ERK1/2-dependent signaling pathway. Suramin, Cibacron blue 3GA, and apyrase attenuated hypoxia-induced ERK1/2 activation and Egr-1 expression. We conclude that hypoxia induces ATP release from endothelial cells and fibroblasts and that the activation of P2 purinoceptors is involved in the regulation of DNA synthesis by fibroblasts under hypoxic conditions.

Immunoreactive localisation of P2Y 1 receptors within the rat and human nodose ganglia and rat brainstem: comparison with [α 33P]deoxyadenosine 5′-triphosphate autoradiography

The present study employed standard peroxidase immunohistochemistry to map the distribution of P2Y 1 receptors in the rat brainstem and nodose ganglia and characterised the binding profile of [α 33P]dATP. Binding of [α 33P]dATP was fully displaceable by adenosine 5′-triphosphate (ATP), and was found on both human and rat nodose ganglia, and throughout the rat brainstem, including the nucleus tractus solitarius and ventrolateral medulla. [α 33P]dATP binding in the human nodose ganglia was significantly displaced by both 2-methylthio ATP and α,β-methylene ATP, but not by uridine 5′-triphosphate, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, 8,8′-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphtalenetrisulfonic) acid (NF279) or N-ethylcarboxamidoadenosine. [α 33P]dATP binding in the rat nodose ganglia and brainstem was significantly displaced by only 2-methylthio ATP, suggesting that [α 33P]dATP is binding to P2Y receptors in the rat. Binding of [α 33P]dATP was also significantly displaced by α,β-methylene adenosine 5′-diphosphate, suggesting a component of the binding is to endogenous ecto-5′-nucleotidase, however, almost all binding could be displaced by a combination of receptor agonists (2-methylthio ATP, uridine 5′-triphosphate and α,β-methylene ATP), suggesting preferential binding to receptors. Immunoreactivity to P2Y 1 receptor (P2Y 1-IR) exhibited similar distribution patterns to [α 33P]dATP binding, with a clear topographic profile. Particularly dense P2Y 1-IR labeling was evident in cells and fibres of the dorsal vagal complex. Immunolabeling was also present in the dorsal motor nucleus of the vagus and nucleus ambiguus, indicating the possibility of P2Y 1 receptors on vagal efferents. Unilateral vagal ligation was also performed to examine the transport of P2Y 1 receptor, using both immunohistochemistry and [α 33P]dATP autoradiography. Accumulations of both P2Y 1-IR and [α 33P]dATP binding were apparent adjacent to both ligatures, suggesting bi-directional transport of P2Y 1 receptors along the rat vagus nerve. This current study represents the first description of P2Y 1 receptor distribution within the rodent brainstem and nodose ganglion and also characterises [α 33P]dATP binding to P2Y receptors.

Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin–sensitive P2y receptors

AbstractDendritic cells (DCs) are considered the principal initiators of immune response because of their ability to migrate into peripheral tissues and lymphoid organs, process antigens, and activate naive T cells. There is evidence that extracellular nucleotides regulate certain functions of DCs via G-protein–coupled P2Y receptors (P2YR) and ion-channel–gated P2X receptors (P2XR). Here we investigated the chemotactic activity and analyzed the migration-associated intracellular signaling events such as actin reorganization and Ca++ transients induced by common P2R agonists such as adenosine 5′-triphosphate (ATP) and 2-methylthioadenosine triphosphate, the P2YR agonists UTP and adenosine 5′-diphosphate (ADP), or the P2XR agonists αβ-methylenadenosine-5′-triphosphate and 2′,3′-(4-benzoyl)benzoyl-ATP. The common P2R agonists and the selective P2YR agonists turned out to be potent chemotactic stimuli for immature DCs, but not for mature DCs. In contrast, P2XR agonists had only marginal chemotactic activity in both DC types. Chemotaxis was paralleled by a rise in the intracellular Ca++concentration and by actin polymerization. Studies with pertussis toxin implicated that intracellular signaling events such as actin polymerization, mobilization of intracellular Ca++, and migration induced by nucleotides was mediated via Gi/oprotein–coupled P2YR. Moreover, functional studies revealed selective down-regulation of this Gi/oprotein–coupled chemotactic P2YR responsiveness during maturation, although immature and mature DCs expressed similar amounts of mRNA for the P2R subtypes (P2Y2R, P2Y4R, P2Y5R, P2Y7R, P2Y11R and P2X1R, P2X4R, P2X7R), and no major differences in respect to the mRNA expression of these receptors could be observed by semiquantitative reverse transcription and polymerase chain reaction (RT-PCR). In summary, our data describe a differential chemotactic response of immature and mature DCs to nucleotides, and lend further support to the hypothesis that P2R are a novel class of immunomodulatory plasma membrane receptors suitable for pharmacological intervention.

Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery.

1. Interactions between A(2A)-adenosine receptors and alpha(2)-, A(1)- and P2- release-inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [(3)H]-noradrenaline, superfused with desipramine-containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz). 2. Blockade of alpha(2)-autoreceptors with yohimbine (1 microM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz. 3. The selective A(2A)-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 1-100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA; 100 nM) or by the P2-receptor agonist 2-methylthioadenosine triphosphate (2-MeSATP; 80 microM). 4. CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304; 30 nM), the A(1)-receptor agonist CPA (100 nM) or the P2-receptor agonist 2-MeSATP (80 microM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow. 5. Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow. 6. It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A(2A)-adenosine receptors is favoured by activation of release inhibitory receptors.

Stimulation of P2 receptors in the ventral tegmental area enhances dopaminergic mechanisms in vivo

It has been shown that endogenous adenosine 5′-triphosphate (ATP) as well as its exogenously applied structural analogue, 2-methylthio ATP (2-MeSATP), facilitate the release of dopamine from axon terminals in the rat nucleus accumbens (NAc) by activating ATP-sensitive P2 receptors. In the present study, reversed microdialysis of 2-MeSATP (10 μM, 100 μM and 1 mM), or its microinjection (0.5, 5.0 and 50 pmol) into the ventral tegmental area (VTA), dose-dependently increased the local extracellular level of dopamine and the locomotion in the open field, respectively. These effects were abolished by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). When applied alone, the antagonist decreased the basal dopamine concentration, indicating that endogenous ATP controls the somatodendritic release of dopamine. Repeated microinjections of 2-MeSATP (5 pmol) once daily for 4 days led to a reproducible locomotor stimulation in the open field. Conditioned locomotion was induced by re-exposure to the novel environment on the seventh day. A challenge with amphetamine (1 mg/kg intraperitoneally) on the eighth day enhanced the locomotor activity in the 2-MeSATP-treated group in the sense of a cross-sensitisation, but failed to do so in the control group. Neurons in the VTA were heavily stained with antibodies developed against the P2Y 1 subtype of P2 receptors. Taken together, our data suggest that P2 receptors (probably of the P2Y 1 subtype) are involved in the initiation of somatodendritic dopamine release in the VTA and thereby may have a profound influence on sensitisation and reward-motivated behaviour.

The effects of P2Y receptor agonists and adenosine on prostaglandin production by the guinea-pig uterus.

1. This study has investigated the effects adenosine 5'-triphosphate (ATP), analogues of ATP, uridine 5'-triphosphate (UTP) and adenosine on prostaglandin output from the guinea-pig uterus superfused in vitro, and from guinea-pig endometrium and myometrium cultured for 24 h. 2. ATP, 2-methylthio ATP and adenosine increased the outputs of prostaglandin F(2 alpha) (PGF(2 alpha)) and 6-keto-PGF(1 alpha) (reflecting PGI(2) production), and UTP increased the output of PGF(2 alpha) from the superfused guinea-pig uterus. These findings support the hypothesis that the contractile effects of ATP, 2-methylthio ATP, UTP and adenosine are mediated by prostaglandins. 3. Suramin (a P2 receptor antagonist) and 8-sulphophenyltheophylline (an A receptor antagonist) blocked the stimulatory actions of ATP and adenosine, respectively, on PGF(2 alpha) output, suggesting that ATP acts on P2 receptors (probably of the P2Y type) and adenosine acts on A receptors in the guinea-pig uterus to increase PGF(2 alpha) production. 4. ATP, 2-methylthio ATP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, UTP and adenosine increased the output of PGF(2 alpha) from guinea-pig endometrium and myometrium after 24 h of culture, with a greater stimulatory effect being exerted on the endometrium than on the myometrium. Little or no stimulatory effect was seen after 2 and 8 h of culture. In addition the effects of ATP, ATP analogues, UTP and adenosine on the outputs of PGE(2) and 6-keto-PGF(1 alpha) from cultured endometrium and myometrium were more variable, with both stimulation and inhibition being observed. 5. The stimulatory effects of ATP and adenosine on PGF(2 alpha) output from the endometrium and myometrium were associated with an increase in the prostaglandin synthesizing capacity of both tissues, due probably to an increase in the amount of prostaglandin H synthase present.

Ethanol inhibition of adenosine 5′-triphosphate-activated current in freshly isolated adult rat hippocampal CA1 neurons

The effect of ethanol on current activated by extracellular adenosine 5′-triphosphate (ATP) was studied in freshly isolated adult rat hippocampal CA1 neurons using whole-cell patch-clamp recording. ATP activated an inward current with an EC 50 value of 18 μM. The inward current was also activated by 2-methylthio ATP (2-MeSATP) and α,β-methylene ATP (α,β-MeATP), inhibited by pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and potentiated by Zn 2+. Ethanol inhibited current activated by 10 μM ATP with an IC 50 value of 83 mM in a voltage-independent manner. Ethanol, 100 mM, shifted the ATP concentration-response curve to the right, increasing the EC 50 value for ATP from 18 to 33 μM, but did not reduce the maximal response to ATP. The results suggest that ethanol can inhibit the function of P2X receptors in adult rat hippocampal neurons by decreasing the apparent affinity of the binding site for ATP.

Effects of adenine nucleosides and nucleotides on neuromuscular transmission to the prostatic stroma of the rat.

1. The aim of this study was to investigate the effects of adenine nucleosides and nucleotides on contractility of the smooth muscle of rat prostate gland. 2. Nerve terminals within rat isolated prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine had no effect on baseline smooth muscle tone but concentration-dependently inhibited electrically-evoked contractile responses. The relative order of potency was ATP congruent with AMP congruent with adenosine>ADP. 3. The inhibition by ATP and adenosine of field stimulation-induced contractions in the rat prostate was antagonized by 8-phenyltheophylline (10 microM), but not by suramin (100 microM) and only slightly by reactive blue 2 (5 microM). 4. The adenosine metabolizing enzyme adenosine deaminase (0.1 unit ml(-1)) inhibited the inhibitory effects of ATP and adenosine. The P2 purinoceptor agonist 2-methylthio ATP (10 nM - 0.1 mM), had no effect on field stimulation-induced contractions of the rat prostate. 5. ATP and adenosine did not modify the contractile responses of the rat prostate to exogenously added noradrenaline (10 microM). 6. Inhibitory concentration-response curves to a number of adenosine analogues with differing stabilities and selectivities for the different adenosine receptors yielded a relative rank order of agonist potency of: N(6)-cyclopentyladenosine (CPA)>N(6)-cyclohexyladenosine (CHA) congruent with (-)-N(6)-(2-phenylisopropyl)-adenosine (R-PIA) congruent with 5'-(N-ethylcarboxamido)-adenosine (NECA)>(+)-N(6)-(2-phenylisopropyl)-adenosine (S-PIA)>2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-ade nosine (CGS 21680). 7. These results indicate that adenine nucleoside and nucleotide induced inhibition of electrically-evoked contractions in the rat prostate occurs through activation of adenosine but not ATP receptors. The relative order of potency of adenosine analogues is consistent with activation of receptors of the A(1)-adenosine receptor subtype. These receptors appear to be prejunctional.

Selective measurement of endothelial or smooth muscle [Ca 2+] i in pressurized/perfused cerebral arteries with fura-2

Despite a critical role for calcium in endothelial regulation of cerebrovascular tone, endothelial intracellular calcium ([Ca 2+] i) has never been measured in the context of an intact pressurized cerebral vessel. The purpose of the present study was to selectively measure endothelial or smooth muscle [Ca 2+] i and diameter in a pressurized/perfused cerebral vessel. In a pressurized rat middle cerebral artery, fura-2 AM was administered selectively to either the luminal (endothelium) or abluminal (smooth muscle) side of the vessel. Selectivity of loading was determined by measuring fura-2 fluorescence before and after removal of the endothelium. Removal of the endothelium virtually eliminated fura-2 fluorescence. In addition, 2-methylthioadenosine triphosphate (2MeS-ATP, a selective endothelial P2 receptor agonist) was used to infer the selectivity of fura-2 loading. It was reasoned that 2MeS-ATP should produce a decrease in smooth muscle [Ca 2+] i and an increase in endothelial [Ca 2+] i in selectively loaded vessels, consistent with its role as an NO-dependent dilator. In smooth muscle loaded vessels, [Ca 2+] i went from 252±8 to 82±9 nM following luminal administration of 2MeS-ATP, whereas in endothelial loaded vessels, [Ca 2+] i went from 137±11 to 271±20 nM. Thus, a method is provided which allows for selective measurement of endothelial or smooth muscle [Ca 2+] i with simultaneous measurement of diameter in a pressurized cerebral vessel.

P2Y receptor regulation of PAI-1 expression in vascular smooth muscle cells.

P2Y-type purine and pyrimidine nucleotide receptors play important roles in the regulation of vascular hemostasis. In this article, the regulation of plasminogen activator inhibitor-1 (PAI-1) expression in rat aortic smooth muscle cells (RASMCs) by adenine and uridine nucleotides was examined and compared. Northern analysis revealed that RASMCs express multiple P2Y receptor subtypes, including P2Y(1), P2Y(2), and P2Y(6). Treatment of RASMCs with UTP increased PAI-1 mRNA expression and extracellular PAI-1 protein levels by 21-fold (P<0.001) and 7-fold (P<0.001), respectively. The ED(50) for the effect of UTP on PAI-1 expression was approximately 1 micromol/L, and its maximal effect occurred at 3 hours. UDP stimulated a 5-fold increase (P<0.005) in PAI-1 expression. In contrast to these potent stimulatory effects of uridine nucleotides, ATP and 2-methylthioadenosine triphosphate (2-MeSATP) caused a small and transient increase in PAI-1 mRNA at 1 hour, followed by a rapid decrease to baseline levels. ADP produced only an inhibitory effect, reducing PAI-1 mRNA levels by 63% (P<0.05) at 3 hours. The relative nucleotide potency in stimulating PAI-1 expression is UTP>UDP>ATP=2-MeSATP, consistent with a predominant role of the P2Y(6) receptor. Further studies revealed that exposure of RASMCs to either ATP or ADP for 3 hours inhibited both UTP- and angiotensin II-stimulated PAI-1 expression by up to 90% (P<0.001). Thus, ATP induced a small and transient upregulation of PAI-1 that was followed by a strong inhibition of PAI-1 expression. These results show that extracellular adenine and uridine nucleotides exert potent and opposing effects on vascular PAI-1 expression.

P2X receptors counteract the vasodilatory effects of endothelium derived hyperpolarising factor

Dilatory responses of extracellular nucleotides were examined in the precontracted isolated rat mesenteric artery. Dilatation mediated by endothelium-derived hyperpolarising factor (EDHF) was studied in the presence of Nω-nitro- l-arginine ( l-NOARG) and indomethacin, and was most potently induced by the selective P2Y 1 receptor agonist adenosine 5′- O-thiodiphosphate (ADPβS), while 2-methylthioadenosine triphosphate (2-MeSATP) and adenosine triphosphate (ATP) were almost inactive. However, after P2X receptor desensitisation (with αβ-methylene-adenosine triphosphate, αβ-MeATP), 2-MeSATP and ATP potently stimulated EDHF-mediated dilatation. This can be explained by simultaneous activation of endothelial P2Y receptors that release EDHF, and depolarising P2X receptors on smooth muscle cells. Uridine triphosphate (UTP) also induced potent dilatation, suggesting EDHF release via P2Y 2/P2Y 4 receptors. Uridine diphosphate (UDP) had only minor dilatory effects, and when pretreated with hexokinase it was almost inactive, suggesting a minor role for P2Y 6 receptors. The nitric oxide (NO) mediated dilatation was studied in the presence of charybdotoxin, apamin and indomethacin. ADPβS, 2-MeSATP, ATP and UTP were all potent relaxant agonists suggesting NO release via P2Y 1 and P2Y 2/P2Y 4 receptors, while UDP was much less potent and efficacious. P2X receptor desensitisation had only minor effect on the NO-mediated dilatations. In conclusion, both EDHF and NO-mediated dilatation can be induced by activation of P2Y 1 and P2Y 2/P2Y 4 receptors. P2X receptor stimulation of smooth muscle cells selectively counteracts the dilatory effect of EDHF.