The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells

Recently, it has been shown that ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-alpha and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5'-O-(3-thiotriphosphate) = 2'- and 3'-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.     

P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol trisphosphate in rat hepatocytes

Stimulation of P2-purinergic receptors by ATP resulted in activation of phosphorylase, which was associated with marked production of inositol trisphosphate (Ins-P3), in rat hepatocytes. ATP also inhibited forskolin-induced accumulation of cAMP in the presence of a phosphodiesterase inhibitor. On the contrary, adenosine or AMP never inhibited the cAMP accumulation, but increased hepatocyte cAMP; the stimulation was antagonized by a methylxanthine. Thus, P1-purinergic receptors are linked to adenylate cyclase in a stimulatory fashion in hepatocytes. Various kinds of purine nucleotides stimulating P2-receptors can be divided into two groups on the basis of their relative abilities to stimulate Ins-P3 production and to inhibit cAMP accumulation; the first group including adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, 5-adenylyl imidodiphosphate, GTP, and guanosine 5'-O-(3-thiotriphosphate) has an efficacy similar to that of ATP, and the second group of nucleotides including alpha, beta-methyleneadenosine 5'-triphosphate, beta, gamma-methyleneadenosine 5'-triphosphate (App(CH)2)p), and GDP exerts considerable inhibitory effects on cAMP accumulation, but only slight effects on inositol lipid metabolism. Treatment of hepatocytes with islet-activating protein, pertussis toxin, blocked the nucleotide-induced inhibition of cAMP accumulation, but exerted only a small effect on Ins-P3 production. In membranes prepared from hepatocytes, forskolin-stimulated adenylate cyclase was inhibited by GTP. This GTP-induced inhibition of the enzyme was susceptible to islet-activating protein and dependent on the concentration of ATP (or its derivatives, ATP gamma S or App(CH2)p). It is concluded that there are two types of P2-purinergic receptors: one is linked to adenylate cyclase via an inhibitory guanine nucleotide regulatory protein (Gi) and the other is linked to phospholipase C.     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5'-triphosphate, UTP, and UDP each increased Ca. Basolateral addition of adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), but not ATP, to the perifusing bath increased Ca. IBDU perfusion with ATP-gamma-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.     

32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor

A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a P2Y-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.     

Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides

Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with the idea that P2Y receptor activation increases the rate of exchange of GTP and GTP analogs for GDP on the relevant guanine nucleotide regulatory protein. Once the active enzymic species is formed, hydrolysis of guanine nucleotide reverts the enzyme to the inactive state.     

Rapid up-regulation of P2Y messengers during granulocytic differentiation of HL-60 cells

HL-60 cells are human promyelocytic cells expressing two ATP receptors: the P2Y(2) and P2Y(11) subtypes. Our Northern blotting experiments have shown that P2Y(2) and P2Y(11) messengers were up-regulated in these cells, rapidly and independently of protein synthesis, following treatment with granulocytic differentiating agents such as retinoic acid, dimethylsulfoxide, granulocyte-colony stimulating factor, dibutyryl cyclic AMP and ATP. AR-C67085 and adenosine 5'-O-(3-thiotriphosphate), two potent agonists of the recombinant P2Y(11) receptor, increased intracellular cAMP concentration in HL-60 cells more potently than ATP itself. These observations support the conclusion that the effect of ATP on HL-60 cell differentiation is mediated by the P2Y(11) receptor.     

ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

In airway smooth muscle cells (SMCs) from mouse lung slices, > or =10 microM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After approximately 1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, > or =0.5 microM adenosine 5'-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5'-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha,beta-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y(2) or P2Y(4) receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction.     

Streospecific substitution of oxygen-18 for sulfur in nucleoside phosphorothioates

Reaction of nucleoside phosphorothioates with N-bromosuccinimide in dioxane and H218O leads to the exchange of sulfur for oxygen-18. Using the Sp-isomers of adenosine 5'-O-(1-thiodiphosphate) and adenosine 3',5'-cyclic phosphorothioate, it can be shown by 31P NMR spectroscopy that this reaction proceeds with inversion of configuration yielding the Rp-isomers of [alpha-18O]ADP and [18O]cAMP, respectively. Adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate) are likewise converted to [beta-18O]ATP and [gamma-18O]ATP although the stereochemistry of the former reaction has yet to be evaluated. With very slight modifications this reaction is applicable to all the common bases.     

Modulation of sodium transport in fetal alveolar epithelial cells by oxygen and corticosterone

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP > UDP >or= UTP > adenosine >or= 2-methylthioadenosine-5'-diphosphate, with alpha,beta-methylene adenosine 5'-triphosphate, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, AMP, UMP, and ATPalphaS inactive] and PCR analysis indicate involvement of P2Y2 and P2Y6 receptors. The signal transduction pathway, which, via the P2Y2 receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on Ca2+/calmodulin-dependent protein kinase II and nuclear factor-kappaB, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1beta and interleukin-6, but not tumor necrosis factor-alpha. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells.     

Domain-specific purinergic signaling in polarized rat cholangiocytes

In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.     

Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P2y-receptor action.     

Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.     

Extracellular ATP differentially modulates Toll-like receptor 4-mediated cell survival and death of microglia

The survival and death rates of inflammatory cells directly control their number and are substantially associated with the degree of inflammation. Microglia, key players in neuroinflammation, often cause excessive reactions implicated in neurological diseases. However, the mechanisms that determine microglial fate under pathological conditions remain to be elucidated. Here, we report that activation by lipopolysaccharide (LPS, a Toll-like receptor 4 ligand), an inflammation inducer, primarily promotes survival of microglia, but as its concentration is increased it induces cell death, resulting in decreased cell number. Moreover, extracellular ATP, which is released upon tissue damage, further enhanced the survival induced by a low LPS concentration and the death induced by a high LPS concentration. The survival-promoting effect of ATP was mimicked by non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), and also by the P2X(7) receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, and was suppressed by the P2X(7) antagonists, Brilliant Blue G and A 438079. On the contrary, the death of LPS-activated microglia was not affected by adenosine 5'-O-(3-thiotriphosphate), but enhanced by adenosine, ATP breakdown product. Thus, extracellular ATP modulates microglial survival and death in different ways involving P2X(7) receptor activation and ATP degradation to adenosine, respectively. Such Toll-like receptor 4/purinergic signaling may provide a fine regulatory system of neuroinflammation through modulating the microglial cell number.     

P2Y11 receptors activate adenylyl cyclase and contribute to nucleotide-promoted cAMP formation in MDCK-D(1) cells. A mechanism for nucleotide-mediated autocrine-paracrine regulation.

Extracellular nucleotides activate P2Y receptors, thereby increasing cAMP formation in Madin-Darby canine kidney (MDCK-D(1)) cells, which express P2Y(1), P2Y(2), and P2Y(11) receptors (Post, S. R., Rump, L. C., Zambon, A., Hughes, R. J., Buda, M. D., Jacobson, J. P., Kao, C. C., and Insel, P. A. (1998) J. Biol. Chem. 273, 23093-23097). The cyclooxygenase inhibitor indomethacin (indo) eliminates UTP-promoted cAMP formation (i.e. via P2Y(2) receptors) but only partially blocks ATP-promoted cAMP formation. The latter response is completely blocked by the nonselective P2Y receptor antagonist suramin. We have sought to identify the mechanism for this P2Y receptor-mediated, indo-resistant cAMP formation. The agonist rank order potencies for cAMP formation were: ADP beta S > or = MT-ADP > 2-MT-ATP > ADP, ATP, ATP gamma S > UTP, AMP, adenosine. We found a similar rank order in MDCK-D(1) cells overexpressing cloned green fluorescent protein-tagged P2Y(11) receptors, but the potency of the agonists was enhanced, consistent with a P2Y(11) receptor-mediated effect. cAMP generation by the P2Y(1) and P2Y(11) receptor agonist ADP beta S was not inhibited by several P2Y(1)-selective antagonists (PPADS, A2P5P, and MRS 2179). Forskolin synergistically enhanced cAMP generation in response to ADP beta S or PGE(2), implying that, like PGE(2), ADP beta S activates adenylyl cyclase via G(s), a conclusion supported by results showing ADP beta S and MT-ADP promoted activation of adenylyl cyclase activity in MDCK-D(1) membranes. We conclude that nucleotide-promoted, indo-resistant cAMP formation in MDCK-D(1) cells occurs via G(s)-linked P2Y(11) receptors. These data describing adenylyl cyclase activity via endogenous P2Y(11) receptors define a mechanism by which released nucleotides can increase cAMP in MDCK-D(1) and other P2Y(11)-containing cells.     

ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Galpha(i) have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Galpha(i)-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca(2+) mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Galpha subunits (Galpha(q/i)). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC(50) of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5'-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5'-O-(thiotriphosphate). Thus, SP1999 is a novel, Galpha(i)-linked receptor for ADP.     

Purine and pyrimidine nucleotide-sensitive phospholipase A(2) in ampulla from frog semicircular canal

This study was attempted to characterize pharmacologically the P2Y receptors triggering phospholipase A(2) (PLA(2)) activation in ampulla from frog semicircular canal. A microassay was developed to screen the abilities of UTP analogs to stimulate [(3)H]arachidonic acid release by labeled ampullas. At 26 degrees C UTP induced a dose-dependent and saturable increase of PLA(2) activity (apparent activation constant 1.3 +/- 0.4 microM, Hill coefficient 0.9 +/- 0.2, maximal stimulating factor 2.0 +/- 0.1). The rank order of potency of agonists for PLA(2) activation was UTP > or = UDP > adenosine 5'-O-(2-thiodiphosphate) = adenosine 5'-O-(3-thiotriphosphate) > or = ATP = 2-methylthio-ATP > or = ADP = diadenosine tetraphosphate > or = alpha,beta-methylene-ATP = CTP > 2' and 3'-O-(4-benzoylbenzoyl)-ATP > or = AMP = UMP > uridine and adenosine. UTP- and 2-methylthio-ATP-induced PLA(2) activations were inhibited by U-73122, GF-109203X, and methyl arachidonyl fluorophosphate. Basal activity was stimulated by phorbol ester and epinephrine and reduced by vasotocin, isoproterenol, prostaglandin E(2), cAMP, and forskolin. H-89 restored the cAMP- and forskolin-inhibited PLA(2) activities. Results indicate that P2Y receptor-mediated PLA(2) stimulation requires phopholipase C and protein kinase C activations and basal activity is inhibited by agonist-stimulated cAMP-dependent mechanisms.     

A permissive role of pertussis toxin substrate G-protein in P2-purinergic stimulation of phosphoinositide turnover and arachidonate release in FRTL-5 thyroid cells. Cooperative mechanism of signal transduction systems

Extracellular ATP and other purinergic agonists were found to inhibit cAMP accumulation by depressing adenylate cyclase as an "inhibitory action" and/or to stimulate arachidonate release in association with phospholipase C or A2 activation and Ca2+ mobilization as "stimulatory actions" in FRTL-5 cells. The stimulatory actions of a group of P2-agonists represented by ATP were partially inhibited by the pretreatment of the cells with islet-activating protein (IAP), pertussis toxin, even when an about 41-kDa membrane protein(s) was completely ADP-ribosylated. Only the IAP-sensitive part of the stimulatory actions was antagonized by 1,3-diethyl-8-phenylxanthine (DPX), an adenosine antagonist. GTP and 8-bromoadenosine 5'-triphosphate (Br-ATP) at two to three orders of higher concentrations than ATP also exerted the stimulatory actions, although they were entirely insensitive to both IAP and DPX. Ligand binding experiments with, [35S]ATP gamma S and [3H]DPX showed that ATP occupies both DPX-sensitive and insensitive receptor sites, whereas GTP does only ATP-displaceable DPX-insensitive sites. Thus, lack of sensitivity of GTP action to DPX was associated with its inability to occupy the DPX-sensitive sites. Adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and P1-agonists such as AMP and N6-(L-2-phenylisopropyl-adenosine (PIA) did not show any stimulatory action. Nevertheless, the agonists remarkably enhanced the stimulatory actions of GTP or Br-ATP. Such permissive actions of PIA and others were sensitive to both IAP and DPX, as were shown for a part of the stimulatory actions of ATP as well as the "inhibitory actions" of both PIA and ATP. We conclude that an IAP substrate G-protein(s) which mediates the inhibitory action of purinergic agonists via a DPX-sensitive purinergic receptor(s) may not directly link to the phospholipase C or A2 system but enhance the system which links to a DPX-insensitive P2-receptor, in an indirect or permissive manner.     

Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells

The purinergic regulation of ciliary activity was studied using small, continuously superfused explants of human nasal epithelium. The P2Y(2) purinoceptor (P2Y(2)-R) was identified as the major purinoceptor regulating ciliary beat frequency (CBF); UTP (EC(50) = 4.7 microM), ATP, and adenosine-5'-O-(3-thiotriphosphate) elicited similar maximal responses, approximately twofold over baseline. ATP, however, elicited a post-peak sustained plateau in CBF (1.83 +/- 0.1-fold), whereas the post-peak CBF response to UTP declined over 15 min to a low-level plateau (1.36 +/- 0.16-fold). UDP also stimulated ciliary beating, probably via P2Y(6)-R, with a maximal effect approximately one-half that elicited by P2Y(2)-R stimulation. Not indicated were P2Y(1)-R-, P2Y(4)-R-, or P2Y(11)-R-mediated effects. A(2B)-receptor agonists elicited sustained responses in CBF approximately equal to those from UTP/ATP [5'-(N-ethylcarboxamido)adenosine, EC(50) = 0.09 microM; adenosine, EC(50) = 0.7 microM]. Surprisingly, ADP elicited a sustained stimulation in CBF. The ADP effect and the post-peak sustained portion of the ATP response in CBF were inhibited by the A(2)-R antagonist 8-(p-sulfophenyl)theophylline. Hence, ATP affects ciliary activity through P2Y(2)-R and, after an apparent ectohydrolysis to adenosine, through A(2B)AR.