T. Kendall Harden

Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of ‘find-me’ signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y2-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y2-/- (also known as P2ry2-/-) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y2-/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y2-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

Towards a revised nomenclature for P1 and P2 receptors

The classification of receptors for adenosine, ATP and ADP (collectively called purinoceptors) has seen a number of developments in the past three years. The important division of receptors into two major classes1 (1) adenosine (P1) receptors and (2) P2 purinoceptors, first suggested by Burnstock in 1978 (Ref.2), has been an abiding one that has set the stage for further subdivision of P2 purinoceptors into P2X and P2Y subtypes on the basis of pharmacological properties3. Later, Dubyak4 summarized the evidence that ATP worked through two different transduction mechanisms: intrinsic ion channels and G protein-coupled receptors. This information, coupled with the cloning of purinoceptors in 1993/94, led Abbracchio and Burnstock5 to propose that purinoceptors should be classified in two families: G protein-coupled receptors termed P2Y purinoceptors, and intrinsic ion channels termed P2X purinoceptors. Developments in recent years have borne out these expectations and a revised nomenclature, essentially adopting the Abbracchio and Burnstock proposal, can now be proposed.

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 (t½;1 ∼;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, [γ-32P]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 × min− 1 × cell− 6). High performance liquid chromatography analysis revealed that the γ-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 [γ-32P]UTP to resting cells resulted in transient formation of [γ-32P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final32P-products of [γ-32P]ATP metabolism were [32P]orthophosphoric acid and a 32P-labeled species that was further purified and identified as [32P]inorganic pyrophosphate. In C6 cells, the formation of [32P]pyrophosphate from [γ-32P]ATP at steady state exceeded by 3-fold that of [32P]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.

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/106cells 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.

Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor

1 . The coding sequence of the P2Y1‐purinoceptor was cloned from a human genomic library. 2 . The open reading frame encodes a protein of 373 amino acids that is 83% identical to the previously cloned chick and turkey P2Y1‐purinoceptor and is ≥95% homologous to the recently cloned rat, mouse, and bovine P2Y1‐purinoceptors. 3 . The human P2Y1‐purinoceptor was stably expressed in 1321N1 human astrocytoma cells using a retroviral vector. Although the P2Y1‐purinoceptor agonist, 2MeSATP, had no effect on inositol phosphate accumulation in 1321N1 cells infected with the control virus, this agonist markedly stimulated inositol phosphate accumulation in cells infected with the P2Y1‐purinoceptor virus. No effect of 2MeSATP on cyclic AMP accumulation was observed in P2Y1‐receptor‐expressing 1321N1 cells. 4 . The pharmacological selectivity of 18 purinoceptor agonists was established for the expressed human P2Y1‐purinoceptor. 2MeSATP was more potent than ATP but less potent than 2MeSADP. ADP also was more potent than ATP. A similar maximal effect was observed with most agonists tested. However, α,β‐MeATP had no effect and 3′‐NH2‐3′‐deoxyATP and A2P4 were partial agonists. The order of potency of agonists for activation of the turkey P2Y1‐purinoceptor, also stably expressed in 1321N1 cells, was identical to that observed for the human P2Y1‐purinoceptor. 5 . C6 glioma cells express a P2Y‐purinoceptor that inhibits adenylyl cyclase but does not activate phospholipase C. Expression of the human P2Y1‐purinoceptor in C6 cells conferred 2MeSATP‐stimulated inositol lipid hydrolysis to these cells. The phospholipase C‐activating human P2Y1‐purinoceptor could be delineated from the endogenous P2Y‐purinoceptor of C6 glioma cells by use of the P2‐purinoceptor antagonist, PPADS, which blocks the P2Y1‐purinoceptor but does not block the endogenous P2Y‐ purinoceptor of C6 cells. P2‐purinoceptor agonists also exhibited differential selectivities for activation of these two P2Y‐purinoceptors.

Pharmacological selectivity of the cloned human P2U-purinoceptor : potent activation by diadenosine tetraphosphate

1 The human P2U‐purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis. 2 High basal levels of inositol phosphates occurred in P2U‐purinoceptor‐expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3 UTP, ATP and ATPγS were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 μM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y‐, P2X‐, and P2T‐purinoceptor agonists, 2MeSATP, α,β‐MeATP, and 2MeSADP, respectively. 4 Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U‐purinoceptor with an EC50 (720 nM) in the range of the most potent P2U‐purinoceptor agonists. AP4A may be a physiologically important activator of P2U‐purinoceptors.

Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate.

The antagonist activity of N6‐methyl 2′‐deoxyadenosine 3′,5′‐bisphosphate (N6MABP) has been examined at the phospholipase C‐coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP‐stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3′,5′‐bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pKB=6.99±0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.

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.

Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors

1 Stimulation of P2Y‐purinoceptors on turkey erythrocytes and many other cell types results in activation of phospholipase C. In contrast, we have observed recently that P2Y‐purinoceptors on C6 rat glioma cells are not coupled to phospholipase C., but rather, inhibit adenylyl cyclase. 2 In this study we investigated the pharmacological selectivity of the P2‐purinoceptor antagonists, suramin, reactive blue 2, and pyridoxal phosphate 6‐azophenyl 2′,4′‐disulphonic acid (PPADS) for phospholipase C‐ and adenylyl cyclase‐coupled P2Y‐purinoceptors. 3 In C6 glioma cells, suramin and reactive blue 2 competitively antagonized the inhibitory effect of 2MeSATP on adenylyl cyclase (pKB = 5.4 ± 0.2 and 7.6 ± 0.1, respectively), whereas PPADS at concentrations up to 100 μm had no effect. 4 In contrast, in the turkey erythrocyte preparation, PPADS at concentrations up to 30 μm was a competitive antagonist of P2Y‐purinoceptor‐stimulated phospholipase C activity (pKB = 5.9 ± 0.1). Suramin and reactive blue 2 produced both a shift to the right of the concentration‐effect of 2MeSATP for the activation of phospholipase C and a significant decrease in the maximal inositol phosphate response. 5 Turkey erythrocytes also express a phospholipase C‐coupled β‐adrenoceptor. Concentrations of PPADS that competitively inhibited the P2Y‐purinoceptor‐mediated response had only minimal effects on the activation of phospholipase C by β‐adrenoceptors. In contrast, suramin and reactive blue 2 produced a non‐competitive inhibition, characterized by decreases in the maximal response to isoprenaline with no change in the potency of this β‐adrenoceptor agonist. 6 The differential effect of PPADS on P2Y‐purinoceptors of C6 glioma cells and turkey erythrocytes adds further support to the idea that different P2Y‐purinoceptor subtypes mediate coupling to adenylyl cyclic and phospholipase C.

Kinetic Scaffolding Mediated by a Phospholipase C–β and Gq Signaling Complex

Reciprocal Regulation An essential step in many signaling cascades is inositol lipid hydrolysis catalyzed by phospholipase C–β. The latter is activated by the α subunit of the heterotrimeric G protein Gq, and it in turn inactivates Gαq, thus sharpening the signal. Waldo et al. (p. 974, published online 21 October) report structural and biochemical data that explain the basis of this reciprocal regulation. One domain of phospholipase C–β binds to activated Gαq. Though the phospholipase C–β active site remains occluded in the structure, the plug is probably removed upon G protein–dependent orientation of the lipase at the membrane. A second domain of phospholipase C–β accelerates guanosine triphosphate hydrolysis by Gαq causing the signaling complex to dissociate. A crystal structure shows how the two components of a central signaling complex regulate each other. Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)–dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide–binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gαq reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein–dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gαq, causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.