Signalling and pharmacological properties of the P2Y receptor.

The P2Y(14) receptor is a relatively broadly expressed G protein-coupled receptor that is prominently associated with immune and inflammatory cells as well as with many epithelia. This receptor historically was thought to be activated selectively by UDP-glucose and other UDP-sugars. However, UDP is also a very potent agonist of this receptor, and may prove to be one of its most important cognate activators.

Extracellular purines are biomarkers of neutrophilic airway inflammation.

Purinergic signalling regulates airway defence mechanisms, suggesting that extracellular purines could serve as airway inflammation biomarkers in cystic fibrosis (CF). The purines adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine were measured in sputum from 21 adults (spontaneously expectorated from seven CF patients, induced from 14 healthy controls) to assess normal values and CF-associated changes. Subsequently, purine levels were measured in bronchoalveolar lavage fluid (BALF) from 37 children (25 CF patients, 12 disease controls) and compared with neutrophil counts, presence of airway infection and lung function. To noninvasively assess airway purines, ATP levels were measured using luminometry in exhaled breath condensate (EBC) from 14 children with CF and 14 healthy controls, then 14 CF children during a pulmonary exacerbation. Both ATP and AMP were elevated in sputum and BALF from CF subjects compared with controls. In BALF, ATP and AMP levels were inversely related to lung function and strongly correlated with neutrophil counts. In EBC, ATP levels were increased in CF relative to controls and decreased after treatment of CF pulmonary exacerbation. The purines adenosine triphosphate and adenosine monophosphate are candidate biomarkers of neutrophilic airways inflammation. Measurement of purines in sputum or exhaled breath condensate may provide a relatively simple and noninvasive method to track this inflammation.

Thrombin-promoted release of UDP-glucose from human astrocytoma cells.

BACKGROUND AND PURPOSE: The P2Y14 receptor is activated by UDP-sugars, most potently by UDP-glucose, but not by free nucleotides, suggesting that UDP-glucose is the cognate agonist for this receptor. However, evidence for regulated release of UDP-glucose is scarce. In the present study, the occurrence of receptor-promoted release of UDP-glucose was investigated, using 1321N1 human astrocytoma cells. EXPERIMENTAL APPROACH: UDP-glucose release and hydrolysis were measured using HPLC-based techniques. Phospholipase C activation and actin cytoskeleton reorganization were assessed by measuring inositol phosphate formation and fluorescence confocal microscopy, respectively. KEY RESULTS: Thrombin and the protease-activating receptor-1 (PAR1) peptide TFLLRNPNDK (PAR1-AP) evoked the release of UDP-glucose and ATP, which was accompanied by enhanced inositol phosphate formation. Although carbachol promoted fourfold greater inositol phosphate formation than thrombin, it failed to promote nucleotide release. Thrombin-promoted nucleotide release was inhibited by BAPTA-AM, brefeldin A and cytochalasin D, and was insensitive to Pertussis toxin and PI3-kinase inhibitors. Thrombin, but not carbachol, induced actin cytoskeleton reorganization, a hallmark of Rho activation in 1321N1 cells. However, PAR-promoted UDP-glucose release was not affected by Rho kinase inhibition. CONCLUSIONS AND IMPLICATIONS: PAR1-evoked UDP-glucose release reflected a Ca2+-dependent mechanism, engaging additional signalling independently of G(i) and Rho kinase activation and requiring a functional actin cytoskeleton and Golgi structures. Our study demonstrates the occurrence of Ca2+ -dependent release of UDP-glucose from astrocytoma cells in response to a physiologically relevant stimulus, that is, a G-protein-coupled receptor agonist. Given the presence of P2Y14 receptors in astrocytes, UDP-glucose may have important autocrine/paracrine functions in the brain.

Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells.

Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl(-) conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A(2B) adenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to "flush" noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.

UTP as an extracellular signaling molecule.

In addition to their central role in many biochemical processes, uridine nucleotides are important extracellular signaling molecules that regulate a broad spectrum of cell functions via activation of P2Y2, P2Y4, and P2Y6 receptors. Cellular release of UTP provides a mechanism for autocrine control of calcium- or protein kinase C-dependent processes.

Cloning and functional characterization of two murine uridine nucleotide receptors reveal a potential target for correcting ion transport deficiency in cystic fibrosis gallbladder.

Extracellular nucleotides regulate transepithelial ion secretion via multiple receptors. The P2Y(2) receptor is the predominant transducer of chloride transport responses to nucleotides in the airways, but the P2 receptors that control ion transport in gastrointestinal epithelia have not been identified. UTP and UDP promote chloride secretion in mouse jejuna and gallbladder epithelia, respectively, and these responses were unaffected by P2Y(2) receptor gene disruption. Pharmacological data suggested the involvement of P2Y(4) and P2Y(6) receptors in gastrointestinal responses. To identify the P2Y receptors responsible for the gastrointestinal actions of UTP and UDP, we have cloned the murine P2Y(4) and P2Y(6) receptors and have stably expressed each in a null cell line to examine the nucleotide-promoted inositol phosphate formation and intracellular Ca(2+) mobilization. The (m)P2Y(4) receptor was potently, but not selectively, activated by UTP (UTP > or = ATP >ITP > GTP > CTP), and it was not activated by UDP or ADP. The (m)P2Y(6) receptor was highly selective for UDP (UDP >> ADP = GDP). The nucleotide selectivities observed with the recombinant (m)P2Y(4) and (m)P2Y(6) receptors resemble those for nucleotide-promoted chloride transport in murine P2Y(2)(-/-) jejuna and gallbladder epithelial cells, respectively. Ion transport responses to nucleotide additions were examined in freshly excised tissues from cystic fibrosis transmembrane regulator-deficient mice. Although the effect of UTP on jejunal short-circuit current (I(sc)) was impaired in the CF mouse, UDP-promoted I(sc) changes were not affected in CF gallbladder epithelium, suggesting that the P2Y(6) receptor is a target for treatment of CF gallbladder disease.

ATP release by mechanically loaded porcine chondrons in pellet culture.

OBJECTIVE: To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures. METHODS: Chondron pellets were formed from 1.6 x 10(6) cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of gamma32P-ATP to resting cultures. RESULTS: Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures. CONCLUSION: The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases.

Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations.

Nucleotides are important extracellular signaling molecules. At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP. Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t(1/2);1 approximately;230-40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration. Although the base-line concentration of ATP remained stable for up to 12 h, [gamma-(32)P]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20-200 fmol x min(-)(1) x cell(-)(6)). High performance liquid chromatography analysis revealed that the gamma-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP. Conversely, addition of [gamma-(32)P]UTP to resting cells resulted in transient formation of [gamma-(32)P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final (32)P-products of [gamma-(32)P]ATP metabolism were [(32)P]orthophosphoric acid and a (32)P-labeled species that was further purified and identified as [(32)P]inorganic pyrophosphate. In C6 cells, the formation of [(32)P]pyrophosphate from [gamma-(32)P]ATP at steady state exceeded by 3-fold that of [(32)P]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides. In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase.

G-protein-coupled receptors as targets for gene transfer vectors using natural small-molecule ligands.

Gene therapy for cystic fibrosis (CF) has focused on correcting electrolyte transport in airway epithelia. However, success has been limited by the failure of vectors to attach and enter into airway epithelia, and may require redirecting vectors to targets on the apical membrane of airway cells that mediate these functions. The G-protein-coupled P2Y2 receptor (P2Y2-R) is abundantly expressed on the airway lumenal surface and internalizes into coated pits upon agonist activation. We tested whether a small-molecule-agonist (UTP) could direct vectors to P2Y2-R and mediate attachment, internalization, and gene transfer. Fluorescein-UTP studies demonstrated that P2Y2-R agonists internalized with their receptor, and biotinylated UTP (BUTP) mediated P2Y2-R-specific internalization of fluorescently labeled streptavidin (SAF) or SAF conjugated to biotinylated Cy3 adenoviral-vector (BCAV). BUTP conjugated to BCAV mediated P2Y2-R-specific gene transfer in (1) adenoviral-resistant A9 and polarized MDCK cells by means of heterologous P2Y2-R, and (2) well-differentiated human airway epithelial cells by means of endogenous P2Y2-R. Targeting vectors with small-molecule-ligands to apical membrane G-protein-coupled receptors may be a feasible approach for successful CF gene therapy.

Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways.

BACKGROUND: Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. MATERIALS AND METHODS: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. RESULTS: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. CONCLUSION: The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.

Nucleotide-regulated calcium signaling in lung fibroblasts and epithelial cells from normal and P2Y(2) receptor (-/-) mice.

To test for the role of the P2Y(2) receptor (P2Y(2)-R) in the regulation of nucleotide-promoted Ca(2+) signaling in the lung, we generated P2Y(2)-R-deficient (P2Y(2)-R(-/-)) mice and measured intracellular Ca(2+)(i) responses (DeltaCa(2+)(i)) to nucleotides in cultured lung fibroblasts and nasal and tracheal epithelial cells from wild type and P2Y(2)-R(-/-) mice. In the wild type fibroblasts, the rank order of potencies for nucleotide-induced DeltaCa(2+)(i) was as follows: UTP >/= ATP >> ADP > UDP. The responses induced by these agonists were completely absent in the P2Y(2)-R(-/-) fibroblasts. Inositol phosphate responses paralleled those of DeltaCa(2+)(i) in both groups. ATP and UTP also induced Ca(2+)(i) responses in wild type airway epithelial cells. In the P2Y(2)-R(-/-) airway epithelial cells, UTP was ineffective. A small fraction (25%) of the ATP response persisted. Adenosine and alpha,beta-methylene ATP were ineffective, and ATP responses were not affected by adenosine deaminase or by removal of extracellular Ca(2+), indicating that neither P1 nor P2X receptors mediated this residual ATP response. In contrast, 2-methylthio-ADP promoted a substantial Ca(2+)(i) response in P2Y(2)-R(-/-) cells, which was inhibited by the P2Y(1) receptor antagonist adenosine 3'-5'-diphosphate. These studies demonstrate that P2Y(2)-R is the dominant purinoceptor in airway epithelial cells, which also express a P2Y(1) receptor, and that the P2Y(2)-R is the sole purinergic receptor subtype mediating nucleotide-induced inositol lipid hydrolysis and Ca(2+) mobilization in mouse lung fibroblasts.

Quantitation of extracellular UTP using a sensitive enzymatic assay.

The wide distribution of the uridine nucleotide-activated P2Y2, P2Y4 and P2Y6 receptors suggests a role for UTP as an important extracellular signalling molecule. However, direct evidence for UTP release and extracellular accumulation has been addressed only recently due to the lack of a sensitive assay for UTP mass. In the present study, we describe a method that is based on the uridinylation of [14C]-glucose-1P by the enzyme UDP-glucose pyrophosphorylase which allows quantification of UTP in the sub-nanomolar concentration range. The UTP-dependent conversion of [14C]-glucose-1P to [14C]-UDP-glucose was made irreversible by including the pyrophosphate scavenger inorganic pyrophosphatase in the reaction medium and [14C]-glucose-1P and [14C]-UDP-glucose were separated and quantified by HPLC. Formation of [14C]-UDP-glucose was linearly observed between 1 and 300 nM UTP. The reaction was highly specific for UTP and was unaffected by a 1000 fold molar excess of ATP over UTP. Release of UTP was measured with a variety of cells including platelets and leukocytes, primary airway epithelial cells, rat astrocytes and several cell lines. In most resting attached cultures, extracellular UTP concentrations were found in the low nanomolar range (1-10 nM in 0.5 ml medium bathing 2.5 cm2 dish). Up to a 20 fold increase in extracellular UTP levels was observed in cells subjected to a medium change. Extracellular UTP levels were 10-30% of the ATP levels in both resting and mechanically-stimulated cultured cells. In unstirred platelets, a 1:100 ratio UTP/ ATP was observed. Extracellular UTP and ATP increased 10 fold in thrombin-stimulated platelets. Detection of UTP in nanomolar concentrations in the medium bathing resting cultures suggests that constitutive release of UTP may provide a mechanism of regulation of the basal activity of uridine nucleotide sensitive receptors.

Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia.

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.

Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation.

ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotides, direct demonstration of the release of cellular UTP has not been reported. Pharmacological studies of the P2Y4 receptor expressed in 1321N1 human astrocytoma cells indicated that this receptor is activated by UTP but not by ATP. Mechanical stimulation of 1321N1 cells also resulted in release of a molecule that markedly activated the expressed P2Y4 receptor. This nucleotide was shown to be UTP by two means. First, high performance liquid chromatography analysis of the medium from [33P]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulation resulted in a large increase in a radioactive species that co-eluted with authentic UTP. This species was degraded by incubation with the nonspecific pyrophosphohydrolase apyrase or with hexokinase and was specifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/10(6) cells within 2 min. This increase was paralleled by a similar augmentation of extracellular ATP levels. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis. Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors.

Identification of an ecto-nucleoside diphosphokinase and its contribution to interconversion of P2 receptor agonists.

The P2Y4 receptor is selectively activated by UTP. Although addition of neither ATP nor UDP alone increased intracellular Ca2+ in 1321N1 human astrocytoma cells stably expressing the P2Y4 receptor, combined addition of these nucleotides resulted in a slowly occurring elevation of Ca2+. The possibility that the stimulatory effect of the combined nucleotides reflected formation of UTP by an extracellular transphosphorylating activity was investigated. Incubation of cells with [3H]UDP or [3H]ADP under conditions in which cellular release of ATP occurred or in the presence of added ATP resulted in rapid formation of the corresponding triphosphates. Transfer of the gamma-phosphate from [gamma-33P]ATP to nucleoside diphosphates confirmed that the extracellular enzymatic activity was contributed by a nucleoside diphosphokinase. The majority of this activity was associated with the cell surface of 1321N1 cells, suggesting involvement of an ectoenzyme. Both ADP and UDP were effective substrates for transphosphorylation. Since ecto-nucleotidase(s) has been considered previously to be the primary enzyme(s) responsible for metabolism of extracellular nucleotides, the relative rates of hydrolysis of ATP, ADP, UTP, and UDP also were determined for 1321N1 cells. All four nucleotides were hydrolyzed with similar Km and Vmax values. Kinetic analyses of the ecto-nucleoside diphosphokinase and ecto-nucleotidase activities indicated that the rate of extracellular transphosphorylation exceeds that of nucleotide hydrolysis by up to 20-fold. Demonstration of the existence of a very active ecto-nucleoside diphosphokinase together with previous observations that stress-induced release of ATP occurs from most cell types indicates that transphosphorylation is physiologically important in the extracellular metabolism of adenine and uridine nucleotides. Since the P2Y receptor class of signaling proteins differs remarkably in their respective specificity for adenine and uridine nucleotides and di- and triphosphates, these results suggest that extracellular interconversion of adenine and uridine nucleotides plays a key role in defining activities in nucleotide-mediated signaling.

UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor.

The presence of the P2Y2 (P(2U)-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl- secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [3H]UTP and [3H]UDP with t1/2 values (at 1 microM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [3H]UDP into [3H]UTP in the presence of ATP. The effects of UDP on [3H]inositol phosphate accumulation, intracellular calcium levels ([Ca2+]i), and Cl- secretory rates (I(Cl-)) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y2 receptor, mucosal addition of UDP promoted [3H]inositol phosphate accumulation with an EC50 of 190 nM. Mucosal addition of UTP stimulated [3H]inositol phosphate accumulation with an EC50 of 280 nM. The maximal effects of mucosal UDP on [3H]inositol phosphate, [Ca2+]i, and I(Cl-) responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [3H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca2+]i and I(Cl-) response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y2 receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.

Pharmacological and second messenger signalling selectivities of cloned P2Y receptors.

1. Four different phospholipase C (PLC)-activating P2Y receptors have been cloned and stably expressed in 1321N1 human astrocytoma cells. These include the human homologues of the P2Y1, P2Y2 and P2Y4 receptors and the rat homologue of the P2Y6 receptor. 2. The nucleotide selectivities of these four receptors have been compared directly by measuring inositol phosphate accumulation in response to nucleotides under conditions in which the initial purity and stability of agonist was rigidly assured and quantitatively assessed. 3. The P2Y1 receptor is specific for adenine nucleotides and slightly more sensitive to disphosphates than triphosphates. When expressed in 1321N1 astrocytoma cells, it couples selectively to the stimulation of PLC and not to the inhibition of adenylyl cyclase. 4. The P2Y2 receptor is activated by UTP and ATP with similar potency and is not activated by nucleoside diphosphates. Diadenosine terraphosphate is a potent agonist at this receptor. 5. The P2Y4 receptor is highly selective for UTP over ATP and is not activated by nucleoside disphosphates. 6. The P2Y6 receptor is activated most potently by UDP, but weakly or not at all by UTP, ADP and ATP. The P2Y6 receptor appears to be identical to the uridine nucleotide-specific receptor previously characterized in C6-2B rat glioma cells. 7. We have identified a P2Y receptor on C6 glioma cells that inhibits adenylyl cyclase but has no effect on PLC. This receptor exhibits a pharmacological selectivity similar but not identical to that of the P2Y1 receptor. When the P2Y1 receptor was expressed in these C6 cells, it conferred an inositol lipid signalling response to adenine nucleotides that was pharmacologically identical to that of the P2Y1 receptor. Thus, the P2Y receptor of C6 glioma cells represents an additional receptor that exhibits the classical pharmacological selectivity of a P2Y1-R, but which couples to adenylyl cyclase rather than to PLC.

Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor.

Observation that the G protein-coupled P2U receptor (P2Y2 receptor) is activated by UTP as well as ATP provided the first indication that a class of uridine nucleotide-responsive receptors might exist. This hypothesis was confirmed by our identification of a uridine nucleotide-specific receptor on C6-2B rat glioma cells and by the recent cloning of two uridine nucleotide-responsive receptors, the P2Y6 receptor [J. Biol. Chem. 270:26152-26158 (1995)] and the P2Y4 receptor [J. Biol. Chem. 270:30849-30852 (1995) and J. Biol. Chem. 270:30845-30848 (1995)]. The relative nucleotide selectivities of these uridine nucleotide-activated receptors have not been established. Therefore, we cloned and expressed the P2Y6 and P2Y4 receptors in 1321N1 human astrocytoma cells and compared their relative selectivities for UDP, UTP, and other uridine and adenine nucleotides with that of the P2Y2 receptor expressed in the same cells. These comparisons were made by measuring inositol phosphate accumulation under conditions in which the initial purity and stability of agonists were rigidly ensured and quantitatively assessed. The data indicate that the P2Y2 receptor is activated with similar potencies by ATP and UTP but not by ADP or UDP; the P2Y6 receptor is activated most potently by UDP but weakly by UTP, ATP, and ADP; and the P2Y4 receptor is activated most potently by UTP, less potently by ATP, and not at all by nucleotide diphosphates. Furthermore, the P2Y6 receptor, which displays a uridine nucleotide selectivity essentially identical to that of the uridine nucleotide-specific receptor in C6-2B cells, was shown to be natively expressed in C6-2B cells and to account for the uridine nucleotide responses originally identified in these cells. These results define the uridine nucleotide selectivity of three phospholipase C-linked receptors: a receptor that is selectively activated by UDP (P2Y6 receptor), selectively activated by UTP (P2Y4 receptor), and activated by UTP and ATP but not by diphosphate nucleotides (P2Y2 receptor).