2Y Receptors Research Articles

P2Y receptors in the gastrointestinal epithelium

AbstractP2Y receptors are widely expressed in all gastrointestinal (GI) epithelia. Commonly, one epithelial tissue or cell expresses several P2Y receptors, which are located in both the apical and basolateral membranes. In almost all studied GI tissues, stimulation of P2Y receptors activates ion secretion, either that of NaCl, KCl, NaHCO3, or KHCO3. In most GI epithelia, an apical P2Y2 receptor is responsible for these ion secretory effects. Major progress during the last 10 years now permits integrated concepts of purinergic regulation of epithelial function. Most importantly, the apical mucus layer can be viewed as a restricted space for local paracrine purinergic regulation of ion transport. Extracellular ATP is present in the mucus in sufficiently high concentrations to activate apical P2Y receptors. Several physiological stimuli have been identified or suggested to trigger ATP release into the luminal space. These include flow‐induced, primary cilium‐dependent ATP secretion into the bile, acidic intraluminal pH in the duodenum, and agonist‐activated secretion of ATP by secretory salivary and pancreatic acini as well as the intestinal crypts. A functional role of the intestinal alkaline phosphatase as ATP cleavage enzyme and thus signal termination is another novel discovery important for duodenal secretion. The overall functional consequence of apical P2Y receptor function is to provide an additional input signal to activate digestive fluid secretion when demanded for by a given physiological stimulus. WIREs Membr Transp Signal 2013, 2:27–36. doi: 10.1002/wmts.74For further resources related to this article, please visit the WIREs website.

Role played by P2X and P2Y receptors in evoking the muscle chemoreflex

The role played by purinergic 2Y receptors in evoking the muscle chemoreflex is not well defined. To shed light on this issue, we compared the pressor responses with popliteal arterial injection of UTP (1 mg/kg), a selective P2Y agonist, with those to popliteal arterial injection of ATP (1 mg/kg), a P2X and P2Y agonist, and to alpha,beta-methylene ATP (50 mug/kg), a selective P2X1 and P2X3 agonist, in decerebrate unanesthetized cats. We found that injection of ATP and alpha,beta-methylene ATP increased mean arterial pressure by 19 +/- 2 and 15 +/- 4 mmHg, whereas UTP had no affect on arterial pressure. In addition, the pressor responses to injection of ATP and alpha,beta-methylene ATP were abolished by section of the sciatic nerve, demonstrating that they were reflex in origin. We conclude that P2Y receptors on thin fiber muscle afferents play no role in evoking the muscle chemoreflex.

Ursodeoxycholic Acid Stimulates Cholangiocyte Fluid Secretion in Mice via CFTR-Dependent ATP Secretion

Cholangiopathies are characterized by impaired cholangiocyte secretion. Ursodeoxycholic acid (UDCA) is widely used for cholangiopathy treatment, but its effects on cholangiocyte secretory functions remain unclear and are the subject of this study. Polarized mouse cholangiocytes in tubular (isolated bile-duct units [IBDU]) or monolayer configuration were obtained from wild-type (WT) and B6-129-Cftr(tm1Kth) and Cftr(tm1Unc) mice that are defective in CFTR, an adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl(-) channel expressed in cholangiocytes. Fluid secretion was assessed by video-optical planimetry, Cl(-) and Ca(2+) efflux by microfluorimetry (6-methoxy-N-ethylquinolinium chloride, fura-2, and fluo-4), adenosine triphosphate (ATP) secretion by luciferin-luciferase assay, and protein kinase C (PKC) by Western blot. UDCA stimulated fluid secretion and Cl(-) efflux in WT-IBDU but not in CFTR-KO-IBDU or in WT-IBDU exposed to CFTR inhibitors. UDCA did not affect intracellular cAMP levels but increased [Ca(2+)]i in WT and not in CFTR-KO cholangiocytes. UDCA stimulated apical ATP secretion in WT but not in CFTR-KO cholangiocytes. UDCA-stimulated [Ca(2+)]i increase was inhibited by suramin, a purinergic 2Y-receptor inhibitor. UDCA stimulated the translocation of PKC-alpha and PKC-epsilon to the plasma membrane. UDCA-stimulated secretion was inhibited by 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and by phospholipase C and PKC inhibitors. UDCA increased ATP output in isolated perfused livers from WT but not from CFTR-KO mice. Our data indicate that UDCA stimulates a CFTR-dependent apical ATP release in cholangiocytes. Secreted ATP activates purinergic 2Y receptors, and, through [Ca(2+)]i increase and PKC activation stimulates Cl(-) efflux and fluid secretion. These data support the concept that CFTR plays a role in modulating purinergic signaling in secretory epithelia and suggest a novel mechanism explaining the choleretic effect of UDCA.

Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca 2+ in several types of cells. We have used Ca 2+-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca 2+ in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca 2+ rise in up to 90% of astrocytes in culture (EC 50 = 7 μM). The NAADP-evoked Ca 2+ rise was maintained in the absence of extracellular Ca 2+, but was suppressed after depleting the Ca 2+ stores of the ER with ATP (20 μM), with cyclopiazonic acid (10 μM) or with ionomycin (5 μM). P 2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′4′-disulfonic acid (PPADS, 100 μM), IP 3 receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 μM) and PLC inhibitor U73122 (10 μM) also reduced or suppressed the NAADP-evoked Ca 2+ rise. NAADP still evoked a Ca 2+ response after application of glycyl- l-phenylalanine-β-naphthylamide (GPN, 200 μM), which permeabilizes lysosomes, or preincubation with H +-ATPase inhibitor bafilomycin A1 (4 μM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 μM), that impairs mitochondrial Ca 2+ handling. In acute brain slices, NAADP (10 μM) evoked Ca 2+ transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca 2+ from inositol 1,4,5-trisphosphate-sensitive Ca 2+ stores in mammalian astrocytes, at least partly by activating metabotropic P 2Y receptors.

Cl−secretion in ATP‐treated renal epithelial C7–MDCK cells is mediated by activation of P2Y1 receptors, phospholipase A2 and protein kinase A

This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.

Adenosine triphosphate inhibits cytokine release from lipopolysaccharide-activated microglia via P 2y receptors

Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor α (TNF-α), interleukin 1β and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10–300 μM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P 2y receptor agonists (ADPβS and 2-methylthio-ATP) on LPS-induced TNF-α release. The rank order of inhibitory potency of endogenous purines on TNF-α release was: ATP>ADP>>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 μM 2-methylthio-ATP showed a potency similar to 100 μM ATP. The addition of ADPβS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P 2y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.

Temporal regulation of agonist efficacy at 5-hydroxytryptamine (5-HT)1A and 5-HT 1B receptors.

Coactivation of purinergic (P 2Y) receptors reduces agonist efficacy at serotonin 1B (5-HT 1B), but not 5-HT 1A receptors. Herein, we report that pretreatment for 5 min with the P 2Y receptor agonist ATP reduced agonist responsiveness at the 5-HT 1A, but not at the 5-HT 1B, receptor. The effect of ATP pretreatment on the 5-HT 1A receptor response rapidly reversed within a 10 min time frame between P 2Y receptor and 5-HT 1A receptor activation. ATP pretreatment effects on 5-HT 1A agonist responsiveness were blocked by the protein kinase inhibitors staurosporine and bisindolylmaleimide, suggesting that the ATP-mediated temporal regulation involves activation of protein kinase C (PKC). Moreover, the temporal effect of ATP was blocked by incubation with 1% ethanol, suggesting that consequences of phospholipase D (PLD) activation play a role. ATP pretreatment blocked the inhibitory effect produced by 5-HT 2C receptor activation on the 5-HT 1A, but not the 5-HT 1B, receptor response, suggesting that the 5-HT 1A receptor itself was the target for PLD/PKC action. Finally, ethanol did not block the reduction in responsiveness of the 5-HT 1A receptor system produced by activation of PKC with phorbol ester treatment, suggesting that PKC activation lies downstream of PLD. Taken together, these data suggest that activation of P 2Y receptors can reduce responsiveness of the 5-HT 1A receptor system via a PLD/PKC-dependent mechanism that is highly dependent upon the temporal pattern of receptor activation. Moreover, this work underscores the importance of time as a variable in receptor signaling cross talk and serves to further illustrate differences between the 5-HT 1A and 5-HT 1B receptor systems.

Purinergic and muscarinic receptor activation activates a common calcium entry pathway in rat neocortical neurons and glial cells

The nature of metabotropic purinergic and muscarinic receptor-mediated increases in intracellular calcium in primary rat neocortical neurons and glial cells has been investigated using fluorescence imaging techniques. Bath-application of ATP and muscarine (10 μM) elicited a characteristic increase in intracellular calcium in both neurons and glial cells. The profile of this response consisted of an initial transient increase followed by a sustained elevation (the plateau phase) which was dependent on extracellular calcium. Examination of the pharmacological basis of the purinergic receptor-mediated calcium response using 10 μM 2-methyl-thio ATP (MeS-ATP) and UTP revealed that P 2Y receptor activation underlies this response. The calcium influx pathway responsible for the sustained calcium response was inhibited by metal ions. In both cell types La 3+ and Zn 2+ (100 μM) effectively inhibited the plateau phase of the response, whilst 100 μM Ni 2+ had little or no effect. In conclusion, P 2Y purinergic and muscarinic receptor activation evoke a sustained increase in intracellular calcium in neocortical neurons and glial cells. This response has similar characteristics to that we have previously described following mGlu 5 activation. We propose that in these cell types stimulation of metabotropic receptors coupled to phosphoinositide turnover activates a common calcium entry pathway that is distinct from voltage-gated calcium channels and resembles store-operated calcium entry.

Extracellular ATP-induced inward current in isolated epithelial cells of the endolymphatic sac

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca 2+ concentration ([Ca 2+] i) in the epithelial cells of the endolymphatic sac were investigated. In zero current-clamp configuration, the average resting membrane potential was −66.8±1.3 mV ( n=18). Application of 30 μM ATP to the bath induced a rapid membrane depolarization by 43.1±2.4 mV ( n=18). In voltage-clamp configuration, ATP-induced inward current at holding potential ( V H) of −60 mV was 169.7±6.3 pA ( n=18). The amplitude of ATP-induced currents increased in sigmoidal fashion over the concentration range between 0.3 and 300 μM with a Hill coefficient ( n) of 1.2 and a dissociation constant ( K d) of 11.7 μM. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP>ATPγs≥ATP>α,β-ATP>ADP=AMP≥adenosine=UTP, was consistent with the properties of the P 2Y receptor. The independence of the reversal potential of the ATP-induced current from Cl − concentration suggests that the current is carried by a cation channel. The relative ionic permeability ratio of the channel modulated by ATP for cations was Ca 2+>Na +>Li +>Ba 2+>Cs +=K +. ATP (10 μM) increased [Ca 2+] i in an external Ca 2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl 2. ATP-induced increase in [Ca 2+] i can be mimicked by application of ionomycin in a Ca 2+-free solution. These results indicate that ATP increases [Ca 2+] i through the P 2Y receptor with a subsequent activation of the non-selective cation channel, and that these effects of ATP are dependent on [Ca 2+] i and extracellular Ca 2+.

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes).

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5-50 microM range (EC50=4.3 microM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 microM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[gamma-S]=2-MeSATP>/=ADP, where ATP[gamma-S] is adenosine 5'-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5'-triphosphate. Adenosine, AMP, alpha, beta-methylene adenosine-5'-triphosphate (alpha,beta-MeATP), beta, gamma methylene adenosine-5'-triphosphate (beta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 microM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 microM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor.

STUDIES ON THE MECHANISMS INVOLVED IN THE ATP-INDUCED RELAXATION IN HUMAN AND RABBIT CORPUS CAVERNOSUM

The effect of ATP in human and rabbit corpus cavernosum (CC) smooth muscle was investigated. Strips of human CC were vertically mounted in an organ bath and the tonic tension was recorded. ATP (0.1-3 mM) induced a concentration-dependent relaxant effect, with a pD 2 value of 3.01 +/− 0.3. The purine-induced relaxation was not affected by L-NAME (100 micro M). In rabbit CC, ATP also induced a concentration-dependent relaxation, which was not influenced by L-NAME or by indomethacin (3 micro M), with a pD 2 value of 3.1 +/− 0.4. The ATP-induced relaxant effect in rabbit CC was increased by both the inhibitor of adenosine reuptake, dipyridamole (3 micro M) and by the inhibitor of adenosine deaminase, EHNA (0.3 micro M). Moreover CGS 15943 (3 micro M), an A 2a adenosine antagonist, reduced the ATP-induced relaxation. UTP was not able to produce relaxation. The two ATP analogues 2-methylthioATP and alpha, beta-methylene ATP were able to induce relaxation in rabbit CC, with the following order of potency: 2-methylthioATP > ATP > alpha, beta-methylene ATP thus suggesting a role for P 2y receptors. However, reactive blue (500 micro M), an unspecific P 2y antagonist, did not modify the ATP relaxant response. The inhibition of phospholipase C by U73122 (3 micro M) and of the endoplasmic reticulum Ca 2+ ATPase by thapsigargin (1 micro M) did not modify the ATP-induced relaxation. The P 2x specific antagonist PPADS (30 micro M) and suramine (500 micro M) were not able to modify the ATP relaxation either in the absence or presence of CGS 15943 (3 micro M). These results confirm that ATP acts as a potent and NO-independent relaxant agent of human and rabbit CC. Our findings also show that the ATP effect is partially attributable to the metabolic breakdown of ATP to adenosine, which acts through A 2a receptor stimulation, but is also due to a direct stimulation of P 2 receptors that are different from the classical P 2y and P 2x receptor subtypes for ATP.

Ligand specificity and ticlopidine effects distinguish three human platelet ADP receptors

Human platelets express adenosine 5′-diphosphate (ADP)-specific purinoceptors of the P 2X and P 2Y receptor superfamily, but their structure, diversity, and precise pharmacological profile is not well understood. Here, functional assays with intact platelets and well-characterized nucleotide derivatives were performed in order to characterize the ligand specificity of these platelet-specific purinoceptors. For the signalling pathways investigated (aggregation, rapid Ca 2+-influx, desensitization of Ca 2+-influx, Ca 2+-mobilization, inhibition of adenylyl cyclase), significant differences in ligand specificity were demonstrated. ADP activated all purinoceptors of human platelets, while adenosine 5′-triphosphate (ATP) was a weak agonist for the P 2X receptor and an antagonist for the P 2Y receptors. The ADP-receptor pathway-antagonist ticlopidine inhibited ADP-evoked aggregation and adenylyl cyclase inhibition but did not affect platelet purinoceptors associated with Ca 2+-influx and Ca 2+-mobilization. These results indicate the presence of three distinct ADP-selective purinoceptors on human platelets.

ATP acting on P 2Y receptors triggers calcium mobilization in Schwann cells at the neuroelectrocyte junction in skate

Schwann cells are integral cellular components of the dense cholinergic presynaptic plexus (nerve plate) which innervates each electrocyte in skate electric organ. Using the Ca 2+-sensitive dye fura-2, we have followed the response in these cells to various chemical challenges. In K + depolarized nerve plates nerve terminals consistently responded with a rapid and sustained Ca 2+ signal. Schwann cell responses to depolarization were rarely seen but, when observed, were always delayed in onset when compared to nerve terminal response (6–10 s later). The possibility that these responses were triggered by mediators released from nerve terminals was tested by direct application of candidate substances. Schwann cells were found to respond to adenosine triphosphate and adenosine diphosphate with a biphasic increase of intracellular Ca 2+ concentration, a rapid peak response being followed in the majority of cells by a sustained plateau phase. In the absence of external Ca 2+ only the transient peak response was observed. Depletion of internal Ca 2+ stores with thapsigargin completely inhibited the adenosine triphosphate-stimulated rise in Schwann cell Ca 2+. The response to adenosine triphosphate was concentration-dependent (EC 50 2.8 μM) and was reversibly blocked by two antagonists of P 2 purinoceptors: suramin and reactive blue 2. Adenosine diphosphate and 2-methylthio-adenosine triphosphate were equipotent with adenosine triphosphate and at high concentrations (100 μM) diadenosine tetraphosphate produced responses comparable to low concentrations of adenosine triphosphate. Adenosine, adenosine monophosphate, the α β-methylene analogues of adenosine triphosphate and adenosine diphosphate, uridine triphosphate, cytidine triphosphate and guanosine triphosphate were without significant effect. These results show that, in skate electric organ Schwann cells, the release of Ca 2+ from intracellular stores is triggered by adenosine triphosphate acting on P 2Y receptors and suggest that Schwann cells may be targets for synaptically-released adenosine triphosphate in the electric organ model of the neuromuscular junction.

Prostanoid secretion by rat hepatic sinusoidal endothelial cells and its regulation by exogenous adenosine triphosphate

We investigated the secretory profiles of prostanoids in two types of nonparenchymal cell from the rat liver, sinusoidal endothelial cells and Kupffer cells, in primary culture both under basal conditions and after stimulation with adenine nucleotides. Prostaglandin (PG) E 2 was the main prostanoid secreted by both types of hepatic nonparenchymal cell in the basal and adenosine triphosphate (ATP)-stimulated states. Time- and concentration-dependent effects of ATP-mediated PGE 2 secretion were noted in sinusoidal endothelial cells, whereas the profile of the relative potencies of individual nucleotides was consistent with the presence of P 2y and P 1 purinergic receptors. In Kupffer cells, the regulation of prostanoid secretion by adenine nucleotides was essentially the same as that in sinusoidal endothelial cells except that adenosine did not stimulate prostanoid secretion and that prostanoid secretion differed somewhat; Kupffer cells secreted relatively more PGF 2α and less 6-keto-PGF 1α than sinusoidal endothelial cells in the presence of ATP, suggesting the presence of only P 2y receptors. In summary, PGE 2 is the main prostanoid secreted by hepatic nonparenchymal cells and its secretion may be stimulated by adenine nucleotides and adenosine.

Adenosine triphosphate and arachidonic acid stimulate glycogenolysis in primary cultures of mouse cerebral cortical astrocytes

Adenosine triphosphate (ATP) promotes glycogenolysis in primary cultures of mouse cerebral cortical astrocytes with an EC 50 of 1.5 μM. A pharmacological analysis indicates an involvement of purinergic P 2Y receptors in this action of ATP Application of either arachidonic acid (AA), or certain unsaturated fatty acids, also results in glycogen breakdown. The EC 50 of AA is approximately 50 μM. Thus ATP and AA can be added to the list of neuroactive agents that control glycogen levels in astrocytes, which includes noradrenaline, vasoactive intestinal peptide (VIP), adenosine and histamine.

Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells

The role of ATP and ADP as intercellular mediators is now well established. The presence of the nucleotides in extracellular fluids can result from several mechanisms: cell lysis, selective permeabilization of the plasma membrane and exocytosis of secretory vesicles, such as platelet dense bodies. Extracellular adenine nucleotides are rapidly degraded by ectonucleotidases expressed inter alia on the surface of endothelial cells. They act on cells via the family of P 2 receptors which encompasses more than 5 subtypes, some of which have been cloned recently. The P 2T, P 2U and P 2Y receptors belong to the superfamily of receptors coupled to G proteins, whereas the P 2X receptor is a cation channel and the P 2Z receptor a non-selective pore. ATP and ADP stimulate the endothelial production of prostacyclin (PGI 2) and nitric oxide (NO), two vasodilators and inhibitors of platelet aggregation, via an increase in cytosolic Ca 2+. This action of adenine nucleotides is believed to limit the extent of intravascular platelet aggregation and to help localize thrombus formation to areas of endothelial damage. The endothelial response to nucleotides is mediated by at least two distinct subtypes of P 2 receptors, P 2Y and P 2U, both coupled to phospholipase C.

Subclasses of purinoceptors in feline bladder

Purines have been shown to inhibit and excite feline detrusor smooth muscle through P 1 and P 2 receptor activation. Several recent studies have demonstrated differences in agonist potency orders for subclasses of purinoceptors, including P 2Y and nucleotide, or P 2U receptors. The current studies were performed to determine the presence of such receptor subtypes in feline detrusor smooth muscle. Cats were surgically prepared for monitoring detrusor smooth muscle contractions as increases in intravesical pressure. Contractions were induced by pelvic nerves stimulation (PNS), ATP, and ATP analogs, such as β, γ-methylene ATP (APPCP), 5′ adenylimido diphosphate (AMP-PNP) and 2-metthylthio (2-MeSATP), ATPγS, UTP, CTP and GTP. These agents all produced contractions and had an agonist potency order of AMP-PNP = APPCP > ATP γS = 2-MeSATP ⪢ ATP > UTP = CTP = GTP. The agonist potency order for inhibition of PNS nerve-evoked bladder contractions was APPCP = AMP-PNP = ATP γS > 2-MeSATP = ATP > UTP = CTP = GTP. Reactive Blue 2 and Coomassie's Brilliant Blue G , two putative P 2Y receptor antagonists, antagonized purine-induced actions. This antagonism and the agonist potency orders suggest the possible presence of novel receptors in detrusor smooth muscle and/or the presence of multiple receptors in detrusor smooth muscle.

Purinergic stimulation of cell division and differentiation: Mechanisms and pharmacological implications

Extracellular purine nucleosides and nucleotides in micromolar concentrations stimulate proliferation of a variety of cell types in vitro and in vivo. As well they act synergistically with NGF to stimulate neurite outgrowth from PC12 cells. A variety of purine nucleosides and deoxyribonucleosides promote cell proliferation and increase intracellular cAMP. Their activities are inhibited by adenosine A 2 receptor antagonists. Only adenosine interacts with the A 2 receptor. We propose that the other nucleosides and deoxyribonucleosides inhibit extracellular adenosine deaminase, thereby increasing the extracellular concentration of adenosine. The nucleotides apparently act by stimulating P 2y receptors coupled to inositol phosphate metabolism. We propose that the nucleosides and nucleotides act synergistically with other growth factors because each has distinct but complementary second messenger systems. If our hypotheses are correct, it should prove possible to modulate the growth and morphogenesis in several cell types using drugs that inhibit or stimulate adenosine A 2 or purine P 2y receptor agonists or the second messenger systems coupled to these receptors.

Adenosine and its nucleotides stimulate proliferation of chick astrocytes and human astrocytoma cells

Aqueous extracts of the brains of 18-day-old white Leghorn chicken embryos contain several substances that stimulate proliferation of primary cultures of chick brain astrocytes. Most of the mitogens are peptides. Purification of one mitogenic fraction was obtained by centrifugation, passage through Amicon Diaflo membranes of nominal molecular weight cutoffs 30, 1 and 0.5 kDa, ion exchange chromatography and reverse phase high performance liquid chromatography (HPLC) using a Deltapak C 18 column. The mitogenic fraction contained to amino acids. On the basis of its behaviour on thin layer chromatography, its ultraviolet absorption spectrum, its 1H and 31P nuclear magnetic resonance spectra and its behaviour on positive and negative ion fast atom bombardment mass spectrometry, the mitogenic material was identified as adenosine-5′-monophosphate (AMP). Other adenosine compounds including adenosine, ADP and ATP also stimulated proliferation of and [ 3H]leucine incorporation into primary cultures of astrocytes. Nitrobenzylthyioinosine (NBTI), an inhibitor of nucleoside transport, did not prevent the stimulation of [ 3H]leucine incorporation into cultured astrocytes. Polyadenylic acid (Poly A), that mimics the effect of adenosine at adenosine receptors, also stimulated proliferation of the astrocytes. The effects of adenosine and Poly A were not inhibited by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) but were inhibited by 1,3-dipropyl-7-methyl-xanthine (DPMX), indicating that adenosine and Poly A acted at the cell surface, likely through adenosine A 2 receptors. The stimulatory effect of ATP was biphasic. The proliferative effect of low, but not of high, concentrations of ATP were abolished by DPMX. The purinergic P 2 receptor agonist 2-methylthioATP and, at higher concentrations, the P 2y agonist, α,β-methyleneATP also stimulated incorporation of [ 3H]thymidine. These data indicate that high concentrations of ATP stimulate cell proliferation through at a P 2, possibly a P 2y receptor. These results have considerable biological significance. After brain injury, or when cells in brain die or become hypoxic, nucleotides and nucleosides are released from the cells. Their extracellular concentrations can exceed those required to stimulate astrocyte proliferation in vitro. Therefore they may be partly responsible for gliotic changes following cell death in brain.

Further subclassification of ATP receptors based on agonist studies

Recent studies using agonist analogues of ATP and other nucleotides have generated some surprising observations which may have ramifications for the classification of P 2 receptors, particularly for those responses currently attributed to P 2Y receptor activation. 2-MethylthioATP (2-MeSATP), the conventional P 2Y receptor agonist, does not interact with ATP in the expected fashion in various models of endothelial function, suggesting that it acts by a different mechanism. Furthermore, in certain cell types where responses to ATP are mediated by phospholipase C activation, 2-MeSATP has little or no activity. Interestingly, the pyrimidine uridine triphosphate (UTP) invariably shows similar protency to ATP in systems where 2-MeSATP is inactive. In this article Steve O'Connor and colleagues discuss these data and their significance, and propose that separate receptors may be responsible; one sensitive to 2-MeSATP and the other, a ‘nucleotide’ receptor, sensitive to UTP.