Update Signal Transduction by Ps-Purinergic Receptors for Extracellular ATP George R. Dubyak Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio
Extracellular adenosine triphosphate (ATP), at micromolar/nanomolar concentrations, has been shown to induce significant functional changes in a wide variety of normal and transformed cell types. While ATP can be nonspecifically released from the cytosol of damaged cells, it is also co-packaged in certain exocytotic vesicles/granules containing conventional neurotransmitters and hormones. The diverse biologic responses to ATP appear to be mediated by a variety of so-called Ps-purinergic, cell surface receptors that are activated upon binding ATP and other nucleotides. Recent physiologic, biochemical, and pharmacologic studies suggest that there are multiple ATP receptor subtypes. These include: (1) G-protein-coupled ATP receptors, which stimulate inositol phospholipid hydrolysis, Ca2+ mobilization, and activation of protein kinase C; (2) ATP receptors that directly activate nonselective cation channels in the plasma membranes of a variety of excitable cell types; and (3) ATP receptors that, via the rapid induction of surface membrane pores permeable to both ions and endogenous metabolites, can produce cytotoxic or activation responses in T lymphocytes and other immune effector cells. In addition to these functional criteria, these putative ATP receptor subtypes can be distinguished by characteristic selectivities for a variety of structurally modified ATP analogs. Current research is directed towards the identification, isolation, and structural characterization of these receptors by both biochemical and molecular biologic approaches.
Extracellular adenosine triphosphate (ATP), at micromolar/nanomolar concentrations, has been shown to induce significant functional changes in a wide variety of normal and transformed cell types. In most cases, these actions of ATP can be functionally distinguished from those elicited in response to occupation of the two well-characterized types (At and A 2) of receptors for adenosine. Thus, a growing body of data suggests the existence of specific cell surface receptors for extracellular ATP. There is a massive pharmacologic literature describing ATP-induced functional responses in various intact organ systems, isolated tissues, and purified cell preparations. This article will focus on four major topics germane to this area: (1)an overview of the signaling roles of extracellular ATP; (2) the classification of putative ATP receptor subtypes; (3) description of recent cell physiologic/biochemical studies aimed at characterization of defined signal transduction pathways that are activated by (Received in original form January 7, 1991 and in revised form February 12, 1991) Address correspondence to: George R. Dubyak, Ph.D., Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106. Abbreviations: adenosine diphosphate, ADP; adenosine triphosphate, ATP; guanine nucleotide-binding regulatory protein, G protein; inositol phospholipid-specific phospholipase C, PI-PLC; uridine triphosphate, UTP. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 295-300, 1991
ATP receptors; and (4) a brief discussion of approaches that have been, or might be, used for the identification, isolation, and structural characterization of these ATP receptor subtypes. The Signaling Role of Extracellular ATP: General Considerations Based on his early and extensive studies on neurotransmission that was resistant to conventional adrenergic and cholinergic antagonists, Burnstock (1) hypothesized that ATP, released at synaptic junctions, might mediate this "nonadrenergic, non-cholinergic" signaling. Nerves releasing ATP as the putative neurotransmitter were termed purinergic. It was further proposed that the target cells of these purinergic nerves expressed cell surface "purinergic" receptors that were activated either by ATP or by the breakdown product, adenosine. The ATP-selective receptors were functionally classified as Pv-purinergic, while the receptors for adenosine were termed Pi-purinergic. Although adenosine receptors are now routinely identified by the At-type and As-type nomenclature, the term Ps-purinergic has persisted as a useful ''umbrella'' classification for putative receptors for ATP. As noted above, the existence of ATP receptors has been postulated due to the wide diversity of organ systems, tissues, and cells that exhibit altered function in response to extracellular ATP. The pharmacologic literature describing
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these various functional responses has been catalogued and summarized in several reviews (2-4), including the proceedings of a recent symposium (4) devoted to discussion of this subject. Particularly significant responses include: (1) stimulation or inhibition of contractility in most vascular and visceral smooth muscle types; (2) stimulation or modulation of the exocytotic release of neurotransmitters or hormones from platelets and multiple endocrine/exocrine tissue types; (3) activation of surfactant release by type II alveolar pneumocytes; (4) stimulation of the release of prostacyclin, endothelium-derived relaxing factor (EDRF), and other vasoactive agents from vascular endothelial cells; and (5) potentiation or "priming" of the functional responses of neutrophils to inflammatory stimuli such as chemotactic peptides and immune complexes. The diversity of these ATP-induced changes in biologic function has prompted both investigation and speculation as to the possible physiologic sources of extracellular ATP. While ATP is present in millimolar concentrations in the cytosol of all eukaryotic cell types, extracellular levels of the nucleotide will normally be maintained at extremely low levels by several mechanisms. First, there is minimal permeation of either ATp4- or MgATp2- (the predominant cytosolic form) across lipid bilayers. Second, ubiquitous ecto-ATPases and ecto-phosphatases rapidly and efficiently hydrolyze extracellular nucleotides (5). Thus, as is the case for any putative signaling agent, appreciable levels of extracellular ATP should occur only transiently and in response to specific physiologic and/or pathologic conditions. Four major sources of extracellular ATP may be considered (reviewed in reference 2). First, it has been demonstrated that ATP is co-packaged in both adrenergic and cholinergic neurotransmitter granules and is released during neurotransmission into synaptic spaces. Second, cytosolic ATP stores can be released by sudden breakage of intact cells, as occurs during rupture of blood vessels and other tissue injury. Third, ATP,·which is also co-packaged with serotonin in platelet granules, has been shown to be released in significant amounts during platelet activation. Finally, ATP has been reported to be released by intact vascular endothelial cells in tissue culture; the mechanism underlying this release has not been defined. These latter three sources (damaged cells, activated platelets, and vascular endothelial cells) suggest that significant amounts of extracellular ATP may locally accumulate at vascular sites of thrombus formation and infection/inflammation, characteristic of inflammatory sites. As previously noted, ATP receptors appear to be expressed by both phagocytic cell types (neutrophils, monocytes, macrophages) and vascular endothelial cells, the vessel elements with which phagocytes interact at vascular lesions. Classification of Cell Surface Receptors Involved in Signal Transduction Research during the past several years has greatly increased our appreciation of the number and diversity of cell surface receptors that activate or attenuate a variety of signal transduction pathways. In particular, the application of molecular biologic methods has facilitated the identification of either cDNA species or genomic DNA species that encode many of these receptors. This structural data, in conjunction with
the results of functional (both physiologic and biochemical) studies, has indicated that signal transducing receptors can be broadly categorized into four major classes: (1) the receptor ion channels; (2) the guanine nucleotide-binding regulatory protein (G-protein)-coupled receptors; (3) the receptor tyrosine kinases; and (4) receptors (e.g., for certain interleukins [6]) exhibiting structural features (or lack thereof) that do not permit their inclusion in the above categories. In general, these latter receptor types, as well as the receptor tyrosine kinases (7), recognize ligands that are large polypeptide growth factors, hormones, or cytokines. In contrast, the receptor ion channels (8, 9) primarily recognize as agonists a host of small organic ligands that function as neurotransmitters; these ligands include acetylcholine, gamma aminobutyric acid (GABA), glutamate, and glycine. As recently reviewed in this journal (lO), G-proteincoupled receptors constitute a large "superfamily" of receptors for many structurally diverse ligands including small organic molecules (e.g., catecholamines and prostaglandins), small (3 to 10 amino acids) peptides (e.g., vasopressin); and larger (20 to 40 amino acids) polypeptide hormones (e.g., glucagon). Given that ATP is a relatively small organic ligand, it is reasonable to postulate that putative ATP receptors are most likely to belong to the family of receptor ion channels and/or the family of G-protein-coupled receptors. As we will discuss below, this hypothesis is indeed consistent with the results of functional studies. Discussion of these functional data may be aided by a brief review of the "common" structural features of receptor ion channels and G-protein-coupled receptors. Functional receptor ion channels (8, 9) exist as a complex of several polypeptide subunits such that the functional domain is formed by the interaction of several discrete subunits to form a channel through which cations or anions can pass with varying degrees of ion selectivity. Such receptors may exist as either homopolymeric or heteropolymeric complexes. The component polypeptide subunits are generally intrinsic membrane proteins that contain multiple transmembrane-spanning domains. Each oligomeric complex forms a channel that stochastically flickers between open and closed states. Ligand binding to one or more subunits alters the kinetics of channel opening. The altered ion conductances then result in changes in the membrane potential of the target cell. The physiologic role of such receptors is the rapid (millisecond time scale) transmission of signals from nerve cell to nerve cell or from nerve cell to muscle cell. G-proteins play an obligatory role in mediating signal transduction by receptors for a broad range of neurotransmitters, hormones, and local mediators. Biochemical and/or molecular biologic analyses indicate that many of these G-protein-coupled receptors may constitute a superfamily of structurally and functionally related membrane proteins (10). The shared structural characteristics of these singlepolypeptide receptors include: an amino-terminal extracellular domain; seven putative transmembrane domains (each consisting of 20 to 25 predominantly hydrophobic amino acids); three intracellular and three extracellular loops that connect the various.transmembrane segments; and an intracellular carboxy-terminus. Two segments within the third intracellular loop appear critical for interaction of the receptors with G-proteins. G-proteins also constitute a super-
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family of related proteins (11). As with G-protein-coupled receptors, the number and diversity of G-proteins and G-protein subunits continue to increase. This diversity underlies the multiplicity of the signal transducing effector systems that are known (or have been hypothesized) to be regulated by G-protein-coupled receptors. These effector systems include adenylate cyclase, a variety of ion channels, and an increasing number of phospholipases. Classification of ATP Receptor Subtypes Pharmacologic classification of receptor subtype (e.g., of the multiple adrenergic receptors) has traditionally involved establishment of the "rank-order of potency;' which characterizes the ability of naturally occurring or synthetic ligands (either agonists or antagonists) to interact with particular receptor subtypes. A similar approach has been applied to the classification of ATP receptors. Although bona fide antagonists of these receptors have yet to be identified or synthesized, various other nucleotides (both natural and synthetic) have been shown to mimic, with different potencies, the ability of ATP to elicit diverse biologic responses. As summarized in an earlier mini-review by Gordon (2), at least three classes of ATP receptors can be grouped on the basis of their selectivities for various nucleotides; these have been termed P 2x- , P 2y - , and P 2Z-type purinergic receptors. As noted in Table 1, these pharmacologically defined classes or subtypes can also be correlated with the functionally defined subtypes that are described below. The only exception is a Ca 2+-mobilizing ATP receptor that can also be activated by low concentrations of uridine triphosphate (UTP) and other non-adenine nucleotides. Coupling of ATP Receptors to Defined Signal Transduction Pathways As previously noted, extracellular ATP elicits diverse responses in many tissues and cell types. Two major lines of research have been used to characterize the general signal transduction systems that underlie the tissue-specific re-
sponses: (1) determination of the intracellular second messengers that change in response to extracellular ATP; and (2) comparison of the ATP-activated changes with those activated in response to occupation of neurotransmitter/hormone receptors belonging to one of the receptor superfamilies described above. Such research has indicated that many of the functional responses to extracellular ATP can be ascribed to, or correlated with, alterations in cellular Ca2+ homeostasis. Utilization of Tsien's fluorescent Ca2+ indicators (12) and appropriate spectrofluorimetric methods have been particularly useful in characterizing ATP-induced signal transduction. Many studies during the last few years have documented that extracellular ATP rapidly (within seconds) increases cytosolic [Ca2+] in the vast majority of cell/tissues that exhibit ATP-induced changes in function. In turn, the ATPinduced increase in cytosolic [Ca2+] can activate a host of secondary signal transduction processes, including: (1) stimulation of phospholipase A2 with consequent production of various eicosanoids; (2) stimulation of diverse Ca2+-dependent regulatory enzymes, including calmodulin-dependent kinases; and (3) activation or inhibition of various plasma membrane ion channels (e.g., Ca-t-activated K+ channels), with consequent changes in membrane potential and excitability. However, comparison of the ATP-induced increases in cytosolic [Ca2+] observed in diverse cell types has shown that these functional responses appear to involve three very different mechanisms of action and thus strongly suggests the existence of at least three distinct ATP receptor subtypes. The diverse mechanisms underlying ATP-induced increases in cytosolic [Ca2+] are outlined in Figure 1 and are discussed in detail below. ATP Receptors Coupled to G-proteins and Inositol Phospholipid-specific Phopholipase C In many cell types, the functional effects elicited by extracellular ATP can be correlated with a rapid ATP-induced mobilization of intracellular Ca 2+ stores (13-17). In such
TABLE 1
Classification of ATP receptor subtypes Signal Transduction Mechanism ATP Receptor Subtype
Tertiary
Secondary
Primary
P2x
Ligand-gated cation (Na, Ca)
Activation of other voltage-gated channels Increased [Ca2+]j
Modulation of membrane potential Activation of: Ca2+-dependent ion channels Ca2+-dependent enzymes
P2y P2u
G-protein activation
Activation of PI-PLC
Increased [Ca2+] Activation of: PLA 2 PC-PLC/PLD Activation of protein kinase C
Activation of other phospholipases (?): PLA 2 PC-PLC/PLD Modulation of G-protein-regulated ion channels P2z
Formation of nonselective pores permeable to ions and metabolites up to 1 kD in mass
Increased [Ca2+] Loss of endogenous nucleotides
Definition of abbreviations: [Ca2+]; = intracellular calcium; PI-PLC phosphatidylcholine-specific phospholipase C/phospholipase D.
=
Activation of Ca2+-dependent enzymes Cytotoxic effects
inositol phospholipid-specific phospholipase C; PLA 2
= phospholipase A2;
PC-PLC
=
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Figure 1. Mechanisms underlyEXTRACELLULAR ATP
"
P2u-receptor
P2y-receptor
j
j
ATP-gated cation channel
ATP-driven pore formation
/
G-protein activation (mul tiple forms)
~
Inositol phospholipidspecific phospholipase C (multiple forms)
Depolarization of plasma _
j
membrane
!
1
vOlt~~e-activated
(1,4,5)InsP3 Production
1
P2z- r e c eptor
P2x-receptor
Ca
Non-selective pores
channel~
/ Increased ca 2+ Influx Across Plasma Membrane
Mobilization of Intracellular ca 2+ stores
~/
INCREASED CYTOSOLIC [ca 2+)
1
ATP-induced Functional Responses (Cell-specific: largely ca 2+-dependent)
cell types, ATP can elicit significant increases in cytosolic [Ca2+] even when the cells are incubated in medium deficient in extracellular Ca2+. Consistent with this CaZ+mobilizing action, extracellular ATP (and other nucleotides) has been shown to trigger rapid activation of inositol phospholipid hydrolysis in such cells. Thus, certain subtypes of ATP receptors are likely to belong to the superfamily of cell surface receptors that are functionally coupled to inositol phospholipid-specific phospholipase C (PI-PLC) effector enzymes (18). Similar to the other receptor types that belong to this superfamily, Ps-purinergic receptors appear to indirectly activate PI-PLC effector enzymes via the mediation of G-proteins. In some, but not all, ATP-responsive cell types, the ability of Ps-purinergic agonists to elicit inositol polyphosphate accumulation is inhibited or attenuated upon pretreatment of the cells with pertussis toxin (16, 17). Fairly extensive agonist selectivity studies have been performed in a number of cell systems in order to define the putative ATP receptor subtype(s) involved in the activation of this particular transmembrane signaling pathway. Such studies suggest that Ps-purinergic activation of PI-PLC/Ca2+ mobilization in various cell types can be further subcategorized into either of two broad nucleotide selectivity groups. In certain cells (e.g., hepatocytes [13] and endothelial cells [14]), adenosine diphosphate (ADP) is equipotent to ATP. In these cells, non-adenine (both purine and pyrimidine) nucleotides are generally inactive or much less potent than ATP/ADP. Conversely, in many other cells (e.g., phagocytic leukocytes [15, 17] and fibroblasts [16]), ADP is several orders of magnitude less potent than ATP in mobilizing intracellular Ca2+ stores. Furthermore, these latter cell types can be activated by relatively low concentrations of certain non-adenine nucleotide triphosphates; in particular, UTP has been shown to be either equipotent to, or more potent than. ATP. In several broken-cell systems (17, 19), the ability of Ps-
ing the increase in cytosolic [Ca2+] activated by different Pzpurinergic receptors for extracellular ATP. Extracellular ATP can interact with several subtypes (u, x, y, z) of Pz-purinergic receptors that can be expressed in different cell types. Occupation of each of these receptor subtypes has been shown to induce rapid increases in cytosolic [Ca2+], with consequent activation of Caz+regulated cellular functions (e.g., contraction in muscle cell types or exocytotic secretion in endocrine/ neuroendocrine cell types). Occupation of both PZY- and Pzu-purinergic receptors primarily induces mobilization of Caz+ sequestered in inositol trisphosphate (1,4,5InsP3 or IP3)-releasable, intracellular stores. Conversely, Pzx- and Pzz-purinergic receptors primarily increase Ca z+ influx, via a variety of channels and "pores," across the plasma membrane.
purinergic agonists to activate PI-PLC is absolutely dependent on the additional presence of guanine nucleotides. Moreover, the ADP analog, P5S]ADP~S, has been successfully used as a radioligand probe of the G-protein-coupled Ps-purinergic receptors expressed in turkey erythrocytes (19). High-affinity binding of this nucleotide to the erythrocyte membranes is inhibited in the presence of guanine nucleotides. The ability of guanine nucleotides to modulate high-affinity binding of receptor agonists is a hallmark of G-protein-coupled receptors (10, 11). These observations strongly suggest the existence of Ps-purinergic receptor subtypes that are likely to be structurally related to the superfamily of G-protein-coupled receptors. As is characteristic of many other classes of receptor agonists, there appear to be multiple subtypes of Ps-purinergic receptors that activate phospholipase C via G-proteins. It will of interest to characterize the nature of the G-proteins that are coupled to these various ATP receptor subtypes. As is true of most PI-PLC-coupled receptors, these particular ATP receptors also activate a variety of other phospholipase-based signal transduction reactions. These not only include phospholipase A z (20) but also phospholipases (both C and D) that selectively hydrolyze phosphatidylcholine (21, 22). Increased activity of these latter enzymes, in concert with the stimulation of PI-PLC, underlies the large ATP-induced increases in diacylglycerol, with consequent activation of protein kinase C, observed in certain cells. These G-protein-coupled ATP receptors may also activate or inhibit other G-protein-regulated signal transduction events. In this regard, Ps-purinergic receptors have been reported to inhibit (via a pertussis toxin-sensitive G-protein) adenylate cyclase activity in glioma cells (23). ATP and UTP have also been shown to inhibit, via a G-protein-mediated mechanism, the so-called voltage-dependent M-current (carried by a specific K+ channel) characteristic of certain amphibian neurons (24). It is likely that many of the diverse bio-
Update
logic responses to ATP observed in particular tissues and cell types will eventually be associated with the activation of these and other G-protein-dependent signal transduction pathways. ATP Receptors that Directly Activate Ion Channels ATP-activated cation conductance pathway(s) appear to be present in the plasma membranes of cardiac myocytes (25), smooth muscle cells (26), sensory neurons (27), pancreatic acinar cells (28), developing myotubes (29), and pheochromocytoma cells (30). The ability of nanomolar to micromolar concentrations of extracellular ATP to activate these conductance pathways has been documented using state-ofthe-art patch-clamp recording of both whole-cell currents and single-channel currents. While there are some differences in the ATP-activated conductance pathways observed in these various cell types, there are several common features. These include: (1) cation specificity with relatively little selectivity for different monovalent cations; (2) substan-: tial activity even at hyperpolarized membrane potentials; (3) sub-second activation and rapid inactivation; (4) an apparent lack of involvement of either G-proteins or diffusible secondmessengers (Ca2+, cyclic adenosine monophosphate [cAMP]) in generating the increased ionic conductances; and (5) a substantial Ca2+ permeability in some cell types. Activation of these ATP-sensitive channels can lead to increased cytosolic [Ca2+] in two ways. In certain cells (e.g., rabbit ear artery [26]), there is sufficient influx of Ca2+ directly via the ATP-activated cation channel to elevate cytosolic [Ca2+]. In other cell types (e.g., cardiomyocytes [31]), the opening 'of. ATP-activated cation channels primarily results in depolarization of the plasma membrane, with secondary activation of voltage-dependent Ca2+ channels. Flux through these latter channels then leads to increased cytosolic [Ca2+]. These data strongly suggest that an ATPactivated cation channel may be a common feature of many excitable cells and may be a likely candidate for explaining at least some aspects of "non-adrenergic, non-cholinergic" neurotransmission. Moreover, the extremely rapid activation of these ionic conductances by ATP suggests that the presumed receptor and the regulated ion channel are parts of a preexisting functional complex. As noted above, this is characteristic of the receptors for a variety of excitatory and inhibitory neurotransmitters. Thus, it is likely that certain ATP receptor subtypes may be structurally related to the superfamily of multi-subunit receptor ion channels. ATP Receptors that Induce Formation of Nonselective Pores There is a long-standing observation that extracellular ATP can effect a "permeabilization" of some cell types to molecules up to 1,000 D in size, including Ca2+, other ions, and endogenous metabolites. Cells in which this response has been extensively characterized include mast cells (32, 33), macrophages (34), and most recently, certain T-lymphocyte subtypes (35). Prolonged opening of these ATP-induced "pores" will lead not only to equilibration of transmembrane ionic gradients (hence, the increase in the intracellular Ca2+) but also to loss of the critical intracellular metabolites (e.g., nucleotides) and eventual cell death. Greenberg and colleagues (34) have utilized this cytotoxic response to
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generate mutant sublines of the J774 mouse macrophage line that are resistant to the cytotoxic effects of extracellular ATP. Recent studies using these mutant cell lines have indicated that this response can be dissociated from the ATPinduced mobilization of intracellular Ca2+ stores discussed above. Thus, the presumed ATP receptors that mediate pore formation appear to be distinct from the G-protein-coupled ATP receptors that activate PI-PLC. Recent patch-clamp electrophysiologic studies of the ion conductance properties of these ATP-induced pores in mast cells indicate that the pores can be functionally distinguished from ATP-activated cation channels also described above (33). Induction of these pores requires the presence of nearmillimolar concentrations of extracellular ATP when cells are incubated in saline containing physiologic concentrations of divalent cations. This contrasts with the activation ofG-protein-coupled ATP receptors or ion channel-coupled ATP receptors by nanomolar to micromolar ATP. For this reason, ATP-induced permeabilization or pore formation has been considered something of a phenomenonologic curiosity with minimal physiologic significance. However, it should be noted that the size of these pores is similar to the gap junction channels that mediate cell-cell coupling. In this regard, Beyer and Steinberg (35) have recently described experiments that indicate that ATP-induced pore formation involves a critical role for the connexin 43 gap junction protein. It is also interesting to note that this ATP-induced pore formation is expressed in cell types (T lymphocytes, mast cells, macrophages) involved in cell-mediated inflammatory or immune responses. Moreover, the ATP-induced permeabilization response is similar to the permeabilization or lytic response triggered by various insect toxins as well as that initiated by cytotoxic T cells. DiVirgilio and associates (36) have reported that cytotoxic T cells and killer cells are themselves highly resistant to the pore-forming and cytotoxic effects of extracellular ATP. Fillipini and colleagues (37) have demonstrated that these same cytotoxic T cells release ATP when incubated with activating ligands such as concanavalin A or monoclonal antibodies against the T-cell antigen receptor. These findings have prompted speculation that ATP may play a physiologic role in cell-mediated immune responses and cytotoxicity. Such models are predicated on the known formation of tight cell-cell junctions between the cytotoxic cells and target cells. ATP, released into the restricted spaces formed by the intercellular junction, might then induce pore formation in the target cells, with resulting loss of intracellular ions and nucleotides. While still speculative and incomplete, this model should provoke interesting experimental approaches in future studies. Future Directions for the Characterization of ATP Receptors It is obvious from the above discussion that the term "putative" is frequently used in reference to Ps-purinergic receptors for ATP. This is necessitated by the fact that virtually all our knowledge concerning the various subtypes of this receptor family is based on functional responses in either intact tissue or isolated cells. Detailed classification of Ps-purinergic subtypes upon such functional responses is limited by the absence of high-affinity selective antagonists.
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Moreover, with several exceptions, there is little ligand binding data of the type usually employed to define subtypes for other well-characterized receptor families (e.g., muscarinic or adrenergic). Given the extremely large number of membrane proteins that recognize ATP as either a substrate or an allosteric ligand, it has proved difficult to develop nucleotide analogs that will specifically (or, at least, primarily) recognize only ATP receptor protein. Nucleotide photoaffinity has been used, with some success, to covalently label possible ATP receptors in vas deferens muscle (38) and turkey erythrocytes (39). When solubilized, affinity-labeled receptors should prove to be a useful starting point for the eventual purification (by conventional protein chemistry) of these particular ATP receptors. Alternatively, expression cloning strategies have been successfully used for the isolation of cDNA species encoding signal transducing receptors in the absence of prior purification of the receptor protein (40). This approach has been particularly useful for the isolation and cloning of several receptors known to belong to the superfamily of G-protein-coupled receptors. Murphy and Tiffany have very recently reported the expression of PI-PLC-coupled ATP/ UTP receptors in Xenopus oocytes injected with mRNA isolated from differentiated HL-60 cells (41). The application of expression and homology cloning methods, coupled with the use of polymerase chain reaction (PCR)-derived probes (41), should prove invaluable for determining the sequence and structure of the various ATP receptor subtypes. Acknowledgments: This review was written during support of the author as an Established Investigator of the American Heart Association.
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A G-protein mutant that inhibits thrombin and Prpurinergic receptor activation of phospholipase A2 • Science 249:662-666. 21. Irving, H. R., and 1. H. Exton. 1987. Phosphatidylcholine breakdown in rat liver plasma membranes: roles of guanine nucleotides and P2purinergic agonists. J. Biol. Chem. 262:3440-3443. 22. Martin, T. W., and K. Michaelis. 1989. P 2-purinergic agonists stimulate phosphodiesteric cleavage of phosphatidylcholine in endothelial cells. Evidence for activation of phospholipase D. J. BioI. Chem. 264: 8847-8856. 23. Pianet, I., M. Merle, and J. Labouesse. 1989. ADP and, indirectly, ATP are potent inhibitors of cAMP production in intact isoproterenolstimulated C6 glioma cells. Biochem. Biophys. Res. Commun. 163: 1150-1157. 24. Brown, D. A. 1988. M-currents. In Ion Channels, Vol. 1. T. Narahashi, editor. Plenum Publishing Corp., New York. 55-94. 25. Friel, D. D., and B. P. Bean. 1988. Two ATP-activated conductances in bullfrog atrial cells. J. Gen. Physiol. 91:1-27. 26. Benham, C. D., and R. W. Tsien. 1987. A novel, receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature 328:275-278. 27. Krishtal, O. A., S. M. Marchenko, A. G. Obukov, and T. M. Volkova. 1988. Receptors for ATP in rat sensory neurons: the structure-function relationship for ligands. Br. J. Pharmacal. 95:1057-1062. 28. Soltoff, S., M. K. McMillian, E. J. Cragoe, Jr., L. C. Cantley, and B. R. Talamo. 1990. Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol. J. Gen. Physiol. 95:319-346. 29. Thomas, S. A., and R. I. Hume. 1990. Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle. J. Gen. Physiol. 95:569-590. 30. Nakazawa, K., K. Fujimori, A. Takanaka, and K. Inoue. 1990. An ATPactivated conductance in pheochromocytoma cells and its suppression by extracellular calcium. J. Physiol. (Lond.). 428:257-272. 31. DeYoung, M. B., and A. Scarpa. 1989. ATP receptor-induced Ca 2+ transients in cardiac myocytes: sources of mobilized Ca 2+. Am. J. Physiol. 257:C750-C758. 32. Cockcroft, S., and B. D. Gomperts. 1980. The ATp4- receptor of rat mast cells. Biochem. J. 188:789-798. 33. Tatham, P. E. R., and M. Lindau. 1990. ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells. J. Gen. Physiol. 95:459-476. 34. Greenberg, S., F. DiVirgilio, T. H. Steinberg, and S. C. Silverstein. 1988. Extracellular nucleotides mediate Ca 2+ fluxes in 1774 macrophages by two distinct mechanisms. J. Biol. Chem. 263:10337-10343. 35. Beyer, E. C., and T. H. Steinberg. 1990. The gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. J. Cell. Bioi. 111: 155a. (Abstr.) 36. DiVirgilio, F., V. Bronte, D. Callavo, andP. Zanovello. 1989. Responses of mouse lymphocytes to extracellular adenosine 5'-triphosphate [ATP]. Lymphocytes with cytotoxic activity are resistant to the permeabilizing effects of ATP. J. Immunol. 143:1955-1960. 37. Fillipini, A., R. E. Thff's, and M. V. Sitkovsky. 1990. Extracellular ATP in T-lymphocyte activation: possible role in effector functions. Proc. Natl. Acad. Sci. USA 87:8267-8271. 38. Fedan,1. S., G. K. Hogaboom, 1. P. O'Donnell, S. 1. Jeng, and R. 1. Guillory. 1985. Interaction of [3H]arylazido aminopropionyl ATP (PH]ANAPP3) with Prpurinergic receptors in the smooth muscle of the isolated guinea-pig vas deferens. Eur. J. Pharmacal. 108:49-61. 39. Boyer, 1. L., C. L. Cooper, and T. K. Harden. 1990. [32P]3 '-O-(4benzoyl)benzoyl adenosine 5'-triphosphate as a photoaffinity label for a phospholipase C-coupled P2y-purinergic receptor. J. Biol. Chem. 265:13515-13520. 40. Snutch, T. P. 1988. The use of Xenopus oocytes to probe synaptic communication. Trends Neurosci. 11:250-256. 41. Murphy, P. M., and H. L. Tiffany. 1990. Characterization of phagocyte P2 nucleotide receptors expressed in Xenopus oocytes. J. Bioi. Chem. 265:11615-11621. 41. Libert, E, M. Parmentier, A. Lefort etal. 1989. Selective amplification and cloning of four new members of the G-protein-coupled receptor family. Science 244:569-572.
Extracellular adenosine triphosphate (ATP), at micromolar/nanomolar concentrations, has been shown to induce significant functional changes in a wide variety of normal and transformed cell types. While ATP can be nonspecifically released from the cytosol of damaged cells, it is also co-packaged in certain exocytotic vesicles/granules containing conventional neurotransmitters and hormones. The diverse biologic responses to ATP appear to be mediated by a variety of so-called Ps-purinergic, cell surface receptors that are activated upon binding ATP and other nucleotides. Recent physiologic, biochemical, and pharmacologic studies suggest that there are multiple ATP receptor subtypes. These include: (1) G-protein-coupled ATP receptors, which stimulate inositol phospholipid hydrolysis, Ca2+ mobilization, and activation of protein kinase C; (2) ATP receptors that directly activate nonselective cation channels in the plasma membranes of a variety of excitable cell types; and (3) ATP receptors that, via the rapid induction of surface membrane pores permeable to both ions and endogenous metabolites, can produce cytotoxic or activation responses in T lymphocytes and other immune effector cells. In addition to these functional criteria, these putative ATP receptor subtypes can be distinguished by characteristic selectivities for a variety of structurally modified ATP analogs. Current research is directed towards the identification, isolation, and structural characterization of these receptors by both biochemical and molecular biologic approaches.
Extracellular adenosine triphosphate (ATP), at micromolar/nanomolar concentrations, has been shown to induce significant functional changes in a wide variety of normal and transformed cell types. In most cases, these actions of ATP can be functionally distinguished from those elicited in response to occupation of the two well-characterized types (At and A 2) of receptors for adenosine. Thus, a growing body of data suggests the existence of specific cell surface receptors for extracellular ATP. There is a massive pharmacologic literature describing ATP-induced functional responses in various intact organ systems, isolated tissues, and purified cell preparations. This article will focus on four major topics germane to this area: (1)an overview of the signaling roles of extracellular ATP; (2) the classification of putative ATP receptor subtypes; (3) description of recent cell physiologic/biochemical studies aimed at characterization of defined signal transduction pathways that are activated by (Received in original form January 7, 1991 and in revised form February 12, 1991) Address correspondence to: George R. Dubyak, Ph.D., Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106. Abbreviations: adenosine diphosphate, ADP; adenosine triphosphate, ATP; guanine nucleotide-binding regulatory protein, G protein; inositol phospholipid-specific phospholipase C, PI-PLC; uridine triphosphate, UTP. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 295-300, 1991
ATP receptors; and (4) a brief discussion of approaches that have been, or might be, used for the identification, isolation, and structural characterization of these ATP receptor subtypes. The Signaling Role of Extracellular ATP: General Considerations Based on his early and extensive studies on neurotransmission that was resistant to conventional adrenergic and cholinergic antagonists, Burnstock (1) hypothesized that ATP, released at synaptic junctions, might mediate this "nonadrenergic, non-cholinergic" signaling. Nerves releasing ATP as the putative neurotransmitter were termed purinergic. It was further proposed that the target cells of these purinergic nerves expressed cell surface "purinergic" receptors that were activated either by ATP or by the breakdown product, adenosine. The ATP-selective receptors were functionally classified as Pv-purinergic, while the receptors for adenosine were termed Pi-purinergic. Although adenosine receptors are now routinely identified by the At-type and As-type nomenclature, the term Ps-purinergic has persisted as a useful ''umbrella'' classification for putative receptors for ATP. As noted above, the existence of ATP receptors has been postulated due to the wide diversity of organ systems, tissues, and cells that exhibit altered function in response to extracellular ATP. The pharmacologic literature describing
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these various functional responses has been catalogued and summarized in several reviews (2-4), including the proceedings of a recent symposium (4) devoted to discussion of this subject. Particularly significant responses include: (1) stimulation or inhibition of contractility in most vascular and visceral smooth muscle types; (2) stimulation or modulation of the exocytotic release of neurotransmitters or hormones from platelets and multiple endocrine/exocrine tissue types; (3) activation of surfactant release by type II alveolar pneumocytes; (4) stimulation of the release of prostacyclin, endothelium-derived relaxing factor (EDRF), and other vasoactive agents from vascular endothelial cells; and (5) potentiation or "priming" of the functional responses of neutrophils to inflammatory stimuli such as chemotactic peptides and immune complexes. The diversity of these ATP-induced changes in biologic function has prompted both investigation and speculation as to the possible physiologic sources of extracellular ATP. While ATP is present in millimolar concentrations in the cytosol of all eukaryotic cell types, extracellular levels of the nucleotide will normally be maintained at extremely low levels by several mechanisms. First, there is minimal permeation of either ATp4- or MgATp2- (the predominant cytosolic form) across lipid bilayers. Second, ubiquitous ecto-ATPases and ecto-phosphatases rapidly and efficiently hydrolyze extracellular nucleotides (5). Thus, as is the case for any putative signaling agent, appreciable levels of extracellular ATP should occur only transiently and in response to specific physiologic and/or pathologic conditions. Four major sources of extracellular ATP may be considered (reviewed in reference 2). First, it has been demonstrated that ATP is co-packaged in both adrenergic and cholinergic neurotransmitter granules and is released during neurotransmission into synaptic spaces. Second, cytosolic ATP stores can be released by sudden breakage of intact cells, as occurs during rupture of blood vessels and other tissue injury. Third, ATP,·which is also co-packaged with serotonin in platelet granules, has been shown to be released in significant amounts during platelet activation. Finally, ATP has been reported to be released by intact vascular endothelial cells in tissue culture; the mechanism underlying this release has not been defined. These latter three sources (damaged cells, activated platelets, and vascular endothelial cells) suggest that significant amounts of extracellular ATP may locally accumulate at vascular sites of thrombus formation and infection/inflammation, characteristic of inflammatory sites. As previously noted, ATP receptors appear to be expressed by both phagocytic cell types (neutrophils, monocytes, macrophages) and vascular endothelial cells, the vessel elements with which phagocytes interact at vascular lesions. Classification of Cell Surface Receptors Involved in Signal Transduction Research during the past several years has greatly increased our appreciation of the number and diversity of cell surface receptors that activate or attenuate a variety of signal transduction pathways. In particular, the application of molecular biologic methods has facilitated the identification of either cDNA species or genomic DNA species that encode many of these receptors. This structural data, in conjunction with
the results of functional (both physiologic and biochemical) studies, has indicated that signal transducing receptors can be broadly categorized into four major classes: (1) the receptor ion channels; (2) the guanine nucleotide-binding regulatory protein (G-protein)-coupled receptors; (3) the receptor tyrosine kinases; and (4) receptors (e.g., for certain interleukins [6]) exhibiting structural features (or lack thereof) that do not permit their inclusion in the above categories. In general, these latter receptor types, as well as the receptor tyrosine kinases (7), recognize ligands that are large polypeptide growth factors, hormones, or cytokines. In contrast, the receptor ion channels (8, 9) primarily recognize as agonists a host of small organic ligands that function as neurotransmitters; these ligands include acetylcholine, gamma aminobutyric acid (GABA), glutamate, and glycine. As recently reviewed in this journal (lO), G-proteincoupled receptors constitute a large "superfamily" of receptors for many structurally diverse ligands including small organic molecules (e.g., catecholamines and prostaglandins), small (3 to 10 amino acids) peptides (e.g., vasopressin); and larger (20 to 40 amino acids) polypeptide hormones (e.g., glucagon). Given that ATP is a relatively small organic ligand, it is reasonable to postulate that putative ATP receptors are most likely to belong to the family of receptor ion channels and/or the family of G-protein-coupled receptors. As we will discuss below, this hypothesis is indeed consistent with the results of functional studies. Discussion of these functional data may be aided by a brief review of the "common" structural features of receptor ion channels and G-protein-coupled receptors. Functional receptor ion channels (8, 9) exist as a complex of several polypeptide subunits such that the functional domain is formed by the interaction of several discrete subunits to form a channel through which cations or anions can pass with varying degrees of ion selectivity. Such receptors may exist as either homopolymeric or heteropolymeric complexes. The component polypeptide subunits are generally intrinsic membrane proteins that contain multiple transmembrane-spanning domains. Each oligomeric complex forms a channel that stochastically flickers between open and closed states. Ligand binding to one or more subunits alters the kinetics of channel opening. The altered ion conductances then result in changes in the membrane potential of the target cell. The physiologic role of such receptors is the rapid (millisecond time scale) transmission of signals from nerve cell to nerve cell or from nerve cell to muscle cell. G-proteins play an obligatory role in mediating signal transduction by receptors for a broad range of neurotransmitters, hormones, and local mediators. Biochemical and/or molecular biologic analyses indicate that many of these G-protein-coupled receptors may constitute a superfamily of structurally and functionally related membrane proteins (10). The shared structural characteristics of these singlepolypeptide receptors include: an amino-terminal extracellular domain; seven putative transmembrane domains (each consisting of 20 to 25 predominantly hydrophobic amino acids); three intracellular and three extracellular loops that connect the various.transmembrane segments; and an intracellular carboxy-terminus. Two segments within the third intracellular loop appear critical for interaction of the receptors with G-proteins. G-proteins also constitute a super-
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family of related proteins (11). As with G-protein-coupled receptors, the number and diversity of G-proteins and G-protein subunits continue to increase. This diversity underlies the multiplicity of the signal transducing effector systems that are known (or have been hypothesized) to be regulated by G-protein-coupled receptors. These effector systems include adenylate cyclase, a variety of ion channels, and an increasing number of phospholipases. Classification of ATP Receptor Subtypes Pharmacologic classification of receptor subtype (e.g., of the multiple adrenergic receptors) has traditionally involved establishment of the "rank-order of potency;' which characterizes the ability of naturally occurring or synthetic ligands (either agonists or antagonists) to interact with particular receptor subtypes. A similar approach has been applied to the classification of ATP receptors. Although bona fide antagonists of these receptors have yet to be identified or synthesized, various other nucleotides (both natural and synthetic) have been shown to mimic, with different potencies, the ability of ATP to elicit diverse biologic responses. As summarized in an earlier mini-review by Gordon (2), at least three classes of ATP receptors can be grouped on the basis of their selectivities for various nucleotides; these have been termed P 2x- , P 2y - , and P 2Z-type purinergic receptors. As noted in Table 1, these pharmacologically defined classes or subtypes can also be correlated with the functionally defined subtypes that are described below. The only exception is a Ca 2+-mobilizing ATP receptor that can also be activated by low concentrations of uridine triphosphate (UTP) and other non-adenine nucleotides. Coupling of ATP Receptors to Defined Signal Transduction Pathways As previously noted, extracellular ATP elicits diverse responses in many tissues and cell types. Two major lines of research have been used to characterize the general signal transduction systems that underlie the tissue-specific re-
sponses: (1) determination of the intracellular second messengers that change in response to extracellular ATP; and (2) comparison of the ATP-activated changes with those activated in response to occupation of neurotransmitter/hormone receptors belonging to one of the receptor superfamilies described above. Such research has indicated that many of the functional responses to extracellular ATP can be ascribed to, or correlated with, alterations in cellular Ca2+ homeostasis. Utilization of Tsien's fluorescent Ca2+ indicators (12) and appropriate spectrofluorimetric methods have been particularly useful in characterizing ATP-induced signal transduction. Many studies during the last few years have documented that extracellular ATP rapidly (within seconds) increases cytosolic [Ca2+] in the vast majority of cell/tissues that exhibit ATP-induced changes in function. In turn, the ATPinduced increase in cytosolic [Ca2+] can activate a host of secondary signal transduction processes, including: (1) stimulation of phospholipase A2 with consequent production of various eicosanoids; (2) stimulation of diverse Ca2+-dependent regulatory enzymes, including calmodulin-dependent kinases; and (3) activation or inhibition of various plasma membrane ion channels (e.g., Ca-t-activated K+ channels), with consequent changes in membrane potential and excitability. However, comparison of the ATP-induced increases in cytosolic [Ca2+] observed in diverse cell types has shown that these functional responses appear to involve three very different mechanisms of action and thus strongly suggests the existence of at least three distinct ATP receptor subtypes. The diverse mechanisms underlying ATP-induced increases in cytosolic [Ca2+] are outlined in Figure 1 and are discussed in detail below. ATP Receptors Coupled to G-proteins and Inositol Phospholipid-specific Phopholipase C In many cell types, the functional effects elicited by extracellular ATP can be correlated with a rapid ATP-induced mobilization of intracellular Ca 2+ stores (13-17). In such
TABLE 1
Classification of ATP receptor subtypes Signal Transduction Mechanism ATP Receptor Subtype
Tertiary
Secondary
Primary
P2x
Ligand-gated cation (Na, Ca)
Activation of other voltage-gated channels Increased [Ca2+]j
Modulation of membrane potential Activation of: Ca2+-dependent ion channels Ca2+-dependent enzymes
P2y P2u
G-protein activation
Activation of PI-PLC
Increased [Ca2+] Activation of: PLA 2 PC-PLC/PLD Activation of protein kinase C
Activation of other phospholipases (?): PLA 2 PC-PLC/PLD Modulation of G-protein-regulated ion channels P2z
Formation of nonselective pores permeable to ions and metabolites up to 1 kD in mass
Increased [Ca2+] Loss of endogenous nucleotides
Definition of abbreviations: [Ca2+]; = intracellular calcium; PI-PLC phosphatidylcholine-specific phospholipase C/phospholipase D.
=
Activation of Ca2+-dependent enzymes Cytotoxic effects
inositol phospholipid-specific phospholipase C; PLA 2
= phospholipase A2;
PC-PLC
=
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Figure 1. Mechanisms underlyEXTRACELLULAR ATP
"
P2u-receptor
P2y-receptor
j
j
ATP-gated cation channel
ATP-driven pore formation
/
G-protein activation (mul tiple forms)
~
Inositol phospholipidspecific phospholipase C (multiple forms)
Depolarization of plasma _
j
membrane
!
1
vOlt~~e-activated
(1,4,5)InsP3 Production
1
P2z- r e c eptor
P2x-receptor
Ca
Non-selective pores
channel~
/ Increased ca 2+ Influx Across Plasma Membrane
Mobilization of Intracellular ca 2+ stores
~/
INCREASED CYTOSOLIC [ca 2+)
1
ATP-induced Functional Responses (Cell-specific: largely ca 2+-dependent)
cell types, ATP can elicit significant increases in cytosolic [Ca2+] even when the cells are incubated in medium deficient in extracellular Ca2+. Consistent with this CaZ+mobilizing action, extracellular ATP (and other nucleotides) has been shown to trigger rapid activation of inositol phospholipid hydrolysis in such cells. Thus, certain subtypes of ATP receptors are likely to belong to the superfamily of cell surface receptors that are functionally coupled to inositol phospholipid-specific phospholipase C (PI-PLC) effector enzymes (18). Similar to the other receptor types that belong to this superfamily, Ps-purinergic receptors appear to indirectly activate PI-PLC effector enzymes via the mediation of G-proteins. In some, but not all, ATP-responsive cell types, the ability of Ps-purinergic agonists to elicit inositol polyphosphate accumulation is inhibited or attenuated upon pretreatment of the cells with pertussis toxin (16, 17). Fairly extensive agonist selectivity studies have been performed in a number of cell systems in order to define the putative ATP receptor subtype(s) involved in the activation of this particular transmembrane signaling pathway. Such studies suggest that Ps-purinergic activation of PI-PLC/Ca2+ mobilization in various cell types can be further subcategorized into either of two broad nucleotide selectivity groups. In certain cells (e.g., hepatocytes [13] and endothelial cells [14]), adenosine diphosphate (ADP) is equipotent to ATP. In these cells, non-adenine (both purine and pyrimidine) nucleotides are generally inactive or much less potent than ATP/ADP. Conversely, in many other cells (e.g., phagocytic leukocytes [15, 17] and fibroblasts [16]), ADP is several orders of magnitude less potent than ATP in mobilizing intracellular Ca2+ stores. Furthermore, these latter cell types can be activated by relatively low concentrations of certain non-adenine nucleotide triphosphates; in particular, UTP has been shown to be either equipotent to, or more potent than. ATP. In several broken-cell systems (17, 19), the ability of Ps-
ing the increase in cytosolic [Ca2+] activated by different Pzpurinergic receptors for extracellular ATP. Extracellular ATP can interact with several subtypes (u, x, y, z) of Pz-purinergic receptors that can be expressed in different cell types. Occupation of each of these receptor subtypes has been shown to induce rapid increases in cytosolic [Ca2+], with consequent activation of Caz+regulated cellular functions (e.g., contraction in muscle cell types or exocytotic secretion in endocrine/ neuroendocrine cell types). Occupation of both PZY- and Pzu-purinergic receptors primarily induces mobilization of Caz+ sequestered in inositol trisphosphate (1,4,5InsP3 or IP3)-releasable, intracellular stores. Conversely, Pzx- and Pzz-purinergic receptors primarily increase Ca z+ influx, via a variety of channels and "pores," across the plasma membrane.
purinergic agonists to activate PI-PLC is absolutely dependent on the additional presence of guanine nucleotides. Moreover, the ADP analog, P5S]ADP~S, has been successfully used as a radioligand probe of the G-protein-coupled Ps-purinergic receptors expressed in turkey erythrocytes (19). High-affinity binding of this nucleotide to the erythrocyte membranes is inhibited in the presence of guanine nucleotides. The ability of guanine nucleotides to modulate high-affinity binding of receptor agonists is a hallmark of G-protein-coupled receptors (10, 11). These observations strongly suggest the existence of Ps-purinergic receptor subtypes that are likely to be structurally related to the superfamily of G-protein-coupled receptors. As is characteristic of many other classes of receptor agonists, there appear to be multiple subtypes of Ps-purinergic receptors that activate phospholipase C via G-proteins. It will of interest to characterize the nature of the G-proteins that are coupled to these various ATP receptor subtypes. As is true of most PI-PLC-coupled receptors, these particular ATP receptors also activate a variety of other phospholipase-based signal transduction reactions. These not only include phospholipase A z (20) but also phospholipases (both C and D) that selectively hydrolyze phosphatidylcholine (21, 22). Increased activity of these latter enzymes, in concert with the stimulation of PI-PLC, underlies the large ATP-induced increases in diacylglycerol, with consequent activation of protein kinase C, observed in certain cells. These G-protein-coupled ATP receptors may also activate or inhibit other G-protein-regulated signal transduction events. In this regard, Ps-purinergic receptors have been reported to inhibit (via a pertussis toxin-sensitive G-protein) adenylate cyclase activity in glioma cells (23). ATP and UTP have also been shown to inhibit, via a G-protein-mediated mechanism, the so-called voltage-dependent M-current (carried by a specific K+ channel) characteristic of certain amphibian neurons (24). It is likely that many of the diverse bio-
Update
logic responses to ATP observed in particular tissues and cell types will eventually be associated with the activation of these and other G-protein-dependent signal transduction pathways. ATP Receptors that Directly Activate Ion Channels ATP-activated cation conductance pathway(s) appear to be present in the plasma membranes of cardiac myocytes (25), smooth muscle cells (26), sensory neurons (27), pancreatic acinar cells (28), developing myotubes (29), and pheochromocytoma cells (30). The ability of nanomolar to micromolar concentrations of extracellular ATP to activate these conductance pathways has been documented using state-ofthe-art patch-clamp recording of both whole-cell currents and single-channel currents. While there are some differences in the ATP-activated conductance pathways observed in these various cell types, there are several common features. These include: (1) cation specificity with relatively little selectivity for different monovalent cations; (2) substan-: tial activity even at hyperpolarized membrane potentials; (3) sub-second activation and rapid inactivation; (4) an apparent lack of involvement of either G-proteins or diffusible secondmessengers (Ca2+, cyclic adenosine monophosphate [cAMP]) in generating the increased ionic conductances; and (5) a substantial Ca2+ permeability in some cell types. Activation of these ATP-sensitive channels can lead to increased cytosolic [Ca2+] in two ways. In certain cells (e.g., rabbit ear artery [26]), there is sufficient influx of Ca2+ directly via the ATP-activated cation channel to elevate cytosolic [Ca2+]. In other cell types (e.g., cardiomyocytes [31]), the opening 'of. ATP-activated cation channels primarily results in depolarization of the plasma membrane, with secondary activation of voltage-dependent Ca2+ channels. Flux through these latter channels then leads to increased cytosolic [Ca2+]. These data strongly suggest that an ATPactivated cation channel may be a common feature of many excitable cells and may be a likely candidate for explaining at least some aspects of "non-adrenergic, non-cholinergic" neurotransmission. Moreover, the extremely rapid activation of these ionic conductances by ATP suggests that the presumed receptor and the regulated ion channel are parts of a preexisting functional complex. As noted above, this is characteristic of the receptors for a variety of excitatory and inhibitory neurotransmitters. Thus, it is likely that certain ATP receptor subtypes may be structurally related to the superfamily of multi-subunit receptor ion channels. ATP Receptors that Induce Formation of Nonselective Pores There is a long-standing observation that extracellular ATP can effect a "permeabilization" of some cell types to molecules up to 1,000 D in size, including Ca2+, other ions, and endogenous metabolites. Cells in which this response has been extensively characterized include mast cells (32, 33), macrophages (34), and most recently, certain T-lymphocyte subtypes (35). Prolonged opening of these ATP-induced "pores" will lead not only to equilibration of transmembrane ionic gradients (hence, the increase in the intracellular Ca2+) but also to loss of the critical intracellular metabolites (e.g., nucleotides) and eventual cell death. Greenberg and colleagues (34) have utilized this cytotoxic response to
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generate mutant sublines of the J774 mouse macrophage line that are resistant to the cytotoxic effects of extracellular ATP. Recent studies using these mutant cell lines have indicated that this response can be dissociated from the ATPinduced mobilization of intracellular Ca2+ stores discussed above. Thus, the presumed ATP receptors that mediate pore formation appear to be distinct from the G-protein-coupled ATP receptors that activate PI-PLC. Recent patch-clamp electrophysiologic studies of the ion conductance properties of these ATP-induced pores in mast cells indicate that the pores can be functionally distinguished from ATP-activated cation channels also described above (33). Induction of these pores requires the presence of nearmillimolar concentrations of extracellular ATP when cells are incubated in saline containing physiologic concentrations of divalent cations. This contrasts with the activation ofG-protein-coupled ATP receptors or ion channel-coupled ATP receptors by nanomolar to micromolar ATP. For this reason, ATP-induced permeabilization or pore formation has been considered something of a phenomenonologic curiosity with minimal physiologic significance. However, it should be noted that the size of these pores is similar to the gap junction channels that mediate cell-cell coupling. In this regard, Beyer and Steinberg (35) have recently described experiments that indicate that ATP-induced pore formation involves a critical role for the connexin 43 gap junction protein. It is also interesting to note that this ATP-induced pore formation is expressed in cell types (T lymphocytes, mast cells, macrophages) involved in cell-mediated inflammatory or immune responses. Moreover, the ATP-induced permeabilization response is similar to the permeabilization or lytic response triggered by various insect toxins as well as that initiated by cytotoxic T cells. DiVirgilio and associates (36) have reported that cytotoxic T cells and killer cells are themselves highly resistant to the pore-forming and cytotoxic effects of extracellular ATP. Fillipini and colleagues (37) have demonstrated that these same cytotoxic T cells release ATP when incubated with activating ligands such as concanavalin A or monoclonal antibodies against the T-cell antigen receptor. These findings have prompted speculation that ATP may play a physiologic role in cell-mediated immune responses and cytotoxicity. Such models are predicated on the known formation of tight cell-cell junctions between the cytotoxic cells and target cells. ATP, released into the restricted spaces formed by the intercellular junction, might then induce pore formation in the target cells, with resulting loss of intracellular ions and nucleotides. While still speculative and incomplete, this model should provoke interesting experimental approaches in future studies. Future Directions for the Characterization of ATP Receptors It is obvious from the above discussion that the term "putative" is frequently used in reference to Ps-purinergic receptors for ATP. This is necessitated by the fact that virtually all our knowledge concerning the various subtypes of this receptor family is based on functional responses in either intact tissue or isolated cells. Detailed classification of Ps-purinergic subtypes upon such functional responses is limited by the absence of high-affinity selective antagonists.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
Moreover, with several exceptions, there is little ligand binding data of the type usually employed to define subtypes for other well-characterized receptor families (e.g., muscarinic or adrenergic). Given the extremely large number of membrane proteins that recognize ATP as either a substrate or an allosteric ligand, it has proved difficult to develop nucleotide analogs that will specifically (or, at least, primarily) recognize only ATP receptor protein. Nucleotide photoaffinity has been used, with some success, to covalently label possible ATP receptors in vas deferens muscle (38) and turkey erythrocytes (39). When solubilized, affinity-labeled receptors should prove to be a useful starting point for the eventual purification (by conventional protein chemistry) of these particular ATP receptors. Alternatively, expression cloning strategies have been successfully used for the isolation of cDNA species encoding signal transducing receptors in the absence of prior purification of the receptor protein (40). This approach has been particularly useful for the isolation and cloning of several receptors known to belong to the superfamily of G-protein-coupled receptors. Murphy and Tiffany have very recently reported the expression of PI-PLC-coupled ATP/ UTP receptors in Xenopus oocytes injected with mRNA isolated from differentiated HL-60 cells (41). The application of expression and homology cloning methods, coupled with the use of polymerase chain reaction (PCR)-derived probes (41), should prove invaluable for determining the sequence and structure of the various ATP receptor subtypes. Acknowledgments: This review was written during support of the author as an Established Investigator of the American Heart Association.
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