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1
Briner, V. A., and F. Kern. "ATP stimulates Ca2+ mobilization by a nucleotide receptor in glomerular endothelial cells." American Journal of Physiology-Renal Physiology 266, no. 2 (February 1, 1994): F210—F217. http://dx.doi.org/10.1152/ajprenal.1994.266.2.f210.
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The present study investigates ATP effects on Ca2+ mobilization in bovine glomerular endothelial cells (GEC) and the receptors mediating ATP response. Extracellular ATP stimulated a rise in inositol 1,4,5-trisphosphate and cytosolic free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. Extracellular Ca2+ depletion did not prevent [Ca2+]i rise. ATP effects were not mediated by P1, P2x, and P2t purinoceptors, since the P1 receptor agonist adenosine and the P2x receptor agonist [alpha,beta-CH2]ATP had no effect on inositol 1-monophosphate (IP) formation and Ca2+ mobilization and ATP does not activate P2t receptors. The P2y receptor antagonist reactive blue (10(-3) M) had little inhibitory effect on ATP (10(-5) M)-stimulated IP formation (15.6 +/- 4.2%) and Ca2+ rise (7.0 +/- 3.0%). According to the classification of purinoceptors, ATP is less potent than 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) in stimulating P2y receptors. In GEC, however, the rank order of potency in stimulating IP and [Ca2+]i rise was ATP > 2-MeS-ATP > ADP. The pyrimidine nucleotide UTP (10(-3) M) induced maximal IP formation (653 +/- 37%) and Ca2+ mobilization (591 +/- 22 nM) similar to ATP (IP 647 +/- 27%; [Ca2+]i 583 +/- 15 nM). At submaximal (10(-5) M) but not at maximal (10(-3) M) doses ATP and UTP effects were additive. ATP and UTP induced specific cross-desensitization. It is concluded that the purinergic nucleotide ATP and pyrimidine nucleotide UTP mediate their effects by a common nucleotide receptor. This receptor differs from P2z and P2y1 receptors, since by definition UTP does not activate these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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Schulze-Lohoff, Eckhard, Christian Hugo, Sylvia Rost, Susanne Arnold, Angela Gruber, Bernhard Brüne, and Ralf Bernd Sterzel. "Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X7receptors." American Journal of Physiology-Renal Physiology 275, no. 6 (December 1, 1998): F962—F971. http://dx.doi.org/10.1152/ajprenal.1998.275.6.f962.
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Mesangial cells undergo cell death both by apoptosis and necrosis during glomerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its receptors may regulate cell death of cultured mesangial cells. Addition of extracellular ATP (300 μM to 5 mM) to cultured rat mesangial cells for 90 min caused a 5.8-fold increase in DNA fragmentation (terminal deoxynucleotidyl transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA fragmentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 expression were reproduced by the purinergic P2Z/P2X7 receptor agonist, 3′- O-(4-benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X7 receptor blocker, oxidized ATP. Transcripts encoding the P2Z/P2X7 receptor were expressed by cultured mesangial cells as determined by Northern blot analysis. P2Z/P2X7 receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X7 receptors by extracellular ATP causes apoptosis and necrosis of cultured mesangial cells. Activation of purinergic P2Z/P2X7 receptors may play a role in causing death of mesangial cells during glomerular disease.
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Schilling, William P., Tanya Wasylyna, George R. Dubyak, Benjamin D. Humphreys, and William G. Sinkins. "Maitotoxin and P2Z/P2X7purinergic receptor stimulation activate a common cytolytic pore." American Journal of Physiology-Cell Physiology 277, no. 4 (October 1, 1999): C766—C776. http://dx.doi.org/10.1152/ajpcell.1999.277.4.c766.
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The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X7ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X7 receptors or 2) MTX and P2Z/P2X7 receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-γ, a maneuver known to upregulate P2Z/P2X7receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X7 receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X7 channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca2+ concentration ([Ca2+]i); the initial increase reflects MTX-induced Ca2+ influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca2+]iand ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X7 receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X7 receptor, MTX-induced increases in [Ca2+]iand ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca2+]ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22°C, a characteristic shared by the P2Z/P2X7-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X7 receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.
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Ecelbarger, C. A., Y. Maeda, C. C. Gibson, and M. A. Knepper. "Extracellular ATP increases intracellular calcium in rat terminal collecting duct via a nucleotide receptor." American Journal of Physiology-Renal Physiology 267, no. 6 (December 1, 1994): F998—F1006. http://dx.doi.org/10.1152/ajprenal.1994.267.6.f998.
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Recent studies in a variety of cell types have revealed several receptor subtypes that bind ATP and trigger increases in intracellular Ca2+ concentration ([Ca2+]i). The present studies were aimed at determining whether similar receptors are present in the rat terminal inner medullary collecting duct (IMCD). [Ca2+]i was measured using fura 2 in tubules dissected from collagenase-treated rat kidneys. ATP (1–100 microM) caused a rapid increase in [Ca2+]i with a prolonged late phase after an initial peak. A similar rise was observed in tubules exposed to UTP or to the poorly hydrolyzable analogue, adenosine 5–-O-(3-thiotriphosphate) (ATP gamma S). In contrast, agonists that bind P2x, P2y, P2z, and P2t purinergic receptors did not affect [Ca2+]i. Removal of extracellular Ca2+ inhibited the response to ATP by approximately 50% with obliteration of the late phase. Furthermore, indomethacin attenuated the rise in [Ca2+]i produced by ATP. Adenosine analogues also increased [Ca2]i apparently by binding to distinct adenosine receptors rather than to the ATP receptor. We conclude that there is a nucleotide receptor in the rat terminal IMCD, which, when occupied, mobilizes intracellular Ca2+.
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Zaborina, Olga, Namita Misra, Jan Kostal, Shilpa Kamath, Vinayak Kapatral, M. El-Azami El-Idrissi, B. S. Prabhakar, and A. M. Chakrabarty. "P2Z-Independent and P2Z Receptor-Mediated Macrophage Killing by Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients." Infection and Immunity 67, no. 10 (October 1, 1999): 5231–42. http://dx.doi.org/10.1128/iai.67.10.5231-5242.1999.
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ABSTRACT We demonstrate that a mucoid, alginate-producing strain ofPseudomonas aeruginosa isolated from the lungs of a cystic fibrosis (CF) patient secretes multiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5′-nucleotidase, and ATP-modifying enzymatic activities. The secretion is triggered at high cell density and in complex media but is greatly reduced when the mucoid cells are grown in mineral salts media or in presence of 5.0 mM Ca2+ or Mg2+. Interestingly, the secretion is triggered primarily in the mucoid CF isolate of strain 8821M (or in strain FRD1) but not in a nonmucoid laboratory strain, PAO1. The purified secreted Ndk shows 100% match in its N-terminal amino acid sequence with that of purified intracellular Ndk and demonstrates similar enzymatic properties. The N-terminal sequence of the purified ATPase isolated from an ndk knockout mutant shows its identity with that of the heat shock chaperonin Hsp60. During fractionation, the flowthrough fraction from a Mono Q column demonstrates the presence of 5′-nucleotidase, adenylate kinase, and a putative ATP reductase activity. These fractions demonstrate high cytotoxic activities for murine peritoneal primary macrophages which can be further stimulated in the presence of ATP or inhibited by pretreatment of macrophages with oxidized ATP (oATP). The cytotoxicity associated with ATP-induced stimulation is believed to be due to activation of macrophage surface-associated P2Z (P2X7) receptors, which are one of the purinergic receptors responsible for pore formation on macrophage membrane. Blocking of these receptors by pretreatment with oATP blocks ATP-induced macrophage cell death. Thus mucoid P. aeruginosa cells elaborate enzymes that modulate the external ATP levels of macrophages, thereby modulating macrophage cell death through P2Z receptor activation. Evidence for the presence of secreted cytotoxic agents that act independently of P2Z receptor activation is also presented.
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Turner, J. T., L. A. landon, S. J. Gibbons, and B. R. Talamo. "Salivary Gland P2 Nucleotide Receptors." Critical Reviews in Oral Biology & Medicine 10, no. 2 (April 1999): 210–24. http://dx.doi.org/10.1177/10454411990100020701.
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The effects of ATP on salivary glands have been recognized since 1982. Functional and pharmacological studies of the P2 nucleotide receptors that mediate the effects of ATP and other extracellular nucleotides have been supported by the cloning of receptor cDNAs, by the expression of the receptor proteins, and by the identification in salivary gland cells of multiple P2 receptor subtypes. Currently, there is evidence obtained from pharmacological and molecular biology approaches for the expression in salivary gland of two P2X ligand-gated ion channels, P2Z/P2X7 and P2X4, and two P2Y G protein-coupled receptors, P2Y1 and P2Y2. Activation of each of these receptor subtypes increases intracellular Ca2+, a second messenger with a key role in the regulation of salivary gland secretion. Through Ca2+ regulation and other mechanisms, P2 receptors appear to regulate salivary cell volume, ion and protein secretion, and increased permeability to small molecules that may be involved in cytotoxicity. Some localization of the various salivary P2 receptor subtypes to specific cells and membrane subdomains has been reported, along with evidence for the co-expression of multiple P2 receptor subtypes within specific salivary acinar or duct cells. However, additional studies in vivo and with intact organ preparations are required to define clearly the roles the various P2 receptor subtypes play in salivary gland physiology and pathology. Opportunities for eventual utilization of these receptors as pharmacotherapeutic targets in diseases involving salivary gland dysfunction appear promising.
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Pérez-Andrés, Encarnación, María Fernández-Rodriguez, Mónica González, Ana Zubiaga, Ainara Vallejo, Itxaso García, Carlos Matute, et al. "Activation of phospholipase D-2 by P2X7 agonists in rat submandibular gland acini." Journal of Lipid Research 43, no. 8 (August 2002): 1244–55. http://dx.doi.org/10.1194/jlr.m100372-jlr200.
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Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca2+ and did not involve intracellular Ca2+ mobilization or phosphoinositide-specific phospholipase C activation. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). PLD activation was fully blocked by the cytosolic phospholipase A2 (PLA2) inhibitor ONO-RS-082 and partially attenuated by the selective Ca2+-dependent cytosolic PLA2 inhibitor, arachidonyl trifluoromethylketone (AACOCF3), or by bromoenol lactone, an inhibitor of Ca2+-independent cytosolic PLA2. Magnesium, which decreases the concentration of ATP4−, and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Using reverse transcription-polymerase chain reaction and Northern blotting techniques, we demonstrated that the PLD isoform stimulated by ATP was PLD-2. Among various ATP analogs, only the P2Z/P2X7 purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. The response to ATP was inhibited by the nonselective P2X purinergic antagonist suramin and by oxidized ATP, a potent P2Z/P2X7 receptor antagonist.It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca2+ influx, cytosolic PLA2, and PKC. Also, the data suggest an involvement of P2X7 receptors in PLD-2 stimulation by ATP.
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Coutinho-Silva, Robson, Pedro M. Persechini, Rodrigo Da Cunha Bisaggio, Jean-Luc Perfettini, Ana Cristina Torres De Sa Neto, Jean M. Kanellopoulos, Iris Motta-Ly, Alice Dautry-Varsat, and David M. Ojcius. "P2Z/P2X7receptor-dependent apoptosis of dendritic cells." American Journal of Physiology-Cell Physiology 276, no. 5 (May 1, 1999): C1139—C1147. http://dx.doi.org/10.1152/ajpcell.1999.276.5.c1139.
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Macrophages and thymocytes express P2Z/P2X7nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Zreceptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis.
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Hickman, SE, J. el Khoury, S. Greenberg, I. Schieren, and SC Silverstein. "P2Z adenosine triphosphate receptor activity in cultured human monocyte- derived macrophages." Blood 84, no. 8 (October 15, 1994): 2452–56. http://dx.doi.org/10.1182/blood.v84.8.2452.2452.
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Abstract The present study shows that human mononuclear phagocytes express a P2Z- like purinergic membrane receptor activity. Extracellular adenosine triphosphate (ATP) induces the formation of nonselective membrane pores in human mononuclear phagocytes that allow the entry of otherwise membrane impermeant fluorescent dyes (YO-PRO-1 or Lucifer yellow) into the cytoplasm of these cells. The percentage of mononuclear phagocytes that was permeabilized by ATP increased as monocytes matured into macrophages. Their response to ATP was inhibited by Mg2+ and oxidized ATP. Benzoylbenzoic-ATP (BzBzATP) was approximately 60% as effective as ATP and adenosine-5 -O-(thiophosphate) (ATP gamma S) was less than 20% as effective as ATP in permeabilizing human macrophages to YO-PRO-1 or Lucifer Yellow. Thus, the human P2Z-like receptor differs from its murine counterpart because BzBzATP, ATP, and ATP gamma S are equally efficacious in permeabilizing murine macrophage-like J774 cells to these dyes. UTP, GTP, and CTP were ineffective in permeabilizing human or murine macrophages to YO-PRO-1. Taken together, these data indicate that human monocyte-derived macrophages express a P2Z-like activity that is pharmacologically distinct from that expressed by their murine counterparts and that expression of these receptors is developmentally regulated in human mononuclear phagocytes.
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Hickman, SE, J. el Khoury, S. Greenberg, I. Schieren, and SC Silverstein. "P2Z adenosine triphosphate receptor activity in cultured human monocyte- derived macrophages." Blood 84, no. 8 (October 15, 1994): 2452–56. http://dx.doi.org/10.1182/blood.v84.8.2452.bloodjournal8482452.
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The present study shows that human mononuclear phagocytes express a P2Z- like purinergic membrane receptor activity. Extracellular adenosine triphosphate (ATP) induces the formation of nonselective membrane pores in human mononuclear phagocytes that allow the entry of otherwise membrane impermeant fluorescent dyes (YO-PRO-1 or Lucifer yellow) into the cytoplasm of these cells. The percentage of mononuclear phagocytes that was permeabilized by ATP increased as monocytes matured into macrophages. Their response to ATP was inhibited by Mg2+ and oxidized ATP. Benzoylbenzoic-ATP (BzBzATP) was approximately 60% as effective as ATP and adenosine-5 -O-(thiophosphate) (ATP gamma S) was less than 20% as effective as ATP in permeabilizing human macrophages to YO-PRO-1 or Lucifer Yellow. Thus, the human P2Z-like receptor differs from its murine counterpart because BzBzATP, ATP, and ATP gamma S are equally efficacious in permeabilizing murine macrophage-like J774 cells to these dyes. UTP, GTP, and CTP were ineffective in permeabilizing human or murine macrophages to YO-PRO-1. Taken together, these data indicate that human monocyte-derived macrophages express a P2Z-like activity that is pharmacologically distinct from that expressed by their murine counterparts and that expression of these receptors is developmentally regulated in human mononuclear phagocytes.
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Ballerini, Patrizia, Michel P. Rathbone, Patrizia Di Iorio, Alessandro Renzetti, Patricia Giuliani, lolanda DʼAlimonte, Oriana Trubiani, Francesco Caciagli, and Renata Ciccarelli. "Rat astroglial P2Z (P2X7) receptors regulate intracellular calcium and purine release." NeuroReport 7, no. 15 (November 1996): 2533–38. http://dx.doi.org/10.1097/00001756-199611040-00026.
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Gu, B., L. J. Bendall, and J. S. Wiley. "Adenosine Triphosphate–Induced Shedding of CD23 and L-Selectin (CD62L) From Lymphocytes Is Mediated by the Same Receptor but Different Metalloproteases." Blood 92, no. 3 (August 1, 1998): 946–51. http://dx.doi.org/10.1182/blood.v92.3.946.
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Abstract CD23 is a transmembrane protein expressed on the surface of B-lymphocytes that binds IgE, CD21, CD11b, and CD11c. High concentrations of soluble CD23 and L-selectin are found in the serum of patients with B-chronic lymphocytic leukemia (B-CLL). Because extracellular adenosine triphosphate (ATP) causes shedding of L-selectin via activation of P2Z/P2X7 receptors expressed on B-CLL lymphocytes, we studied the effect of ATP on shedding of CD23. ATP-induced shedding of CD23 at an initial rate of 12% of that for L-selectin, whereas the EC50 for ATP was identical (35 μmol/L) for shedding of both molecules. Furthermore, benzoylbenzoyl ATP also produced shedding of CD23 and L-selectin with the same agonist EC50 values for both (10 μmol/L). Inactivation of the P2Z/P2X7 receptor by preincubation with oxidized ATP abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2Z/P2X7 receptor, inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nmol/L). Ro 31-9790, a membrane permeant zinc chelator that inhibits the phorbol-ester-stimulated shedding of L-selectin, also inhibited shedding of CD23 from B-CLL lymphocytes. However, the IC50 for this inhibition by Ro31-9790 was different for L-selectin and CD23 (83 v 6 μmol/L, respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester, CD23 was not lost under these conditions. The data show that extracellular ATP induces shedding of L-selectin and CD23 from B-CLL lymphocytes by an action mediated by the P2Z/P2X7 receptor. However, different membrane metalloproteases seem to mediate the shedding of L-selectin and CD23. © 1998 by The American Society of Hematology.
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Gu, B., L. J. Bendall, and J. S. Wiley. "Adenosine Triphosphate–Induced Shedding of CD23 and L-Selectin (CD62L) From Lymphocytes Is Mediated by the Same Receptor but Different Metalloproteases." Blood 92, no. 3 (August 1, 1998): 946–51. http://dx.doi.org/10.1182/blood.v92.3.946.415a24_946_951.
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CD23 is a transmembrane protein expressed on the surface of B-lymphocytes that binds IgE, CD21, CD11b, and CD11c. High concentrations of soluble CD23 and L-selectin are found in the serum of patients with B-chronic lymphocytic leukemia (B-CLL). Because extracellular adenosine triphosphate (ATP) causes shedding of L-selectin via activation of P2Z/P2X7 receptors expressed on B-CLL lymphocytes, we studied the effect of ATP on shedding of CD23. ATP-induced shedding of CD23 at an initial rate of 12% of that for L-selectin, whereas the EC50 for ATP was identical (35 μmol/L) for shedding of both molecules. Furthermore, benzoylbenzoyl ATP also produced shedding of CD23 and L-selectin with the same agonist EC50 values for both (10 μmol/L). Inactivation of the P2Z/P2X7 receptor by preincubation with oxidized ATP abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2Z/P2X7 receptor, inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nmol/L). Ro 31-9790, a membrane permeant zinc chelator that inhibits the phorbol-ester-stimulated shedding of L-selectin, also inhibited shedding of CD23 from B-CLL lymphocytes. However, the IC50 for this inhibition by Ro31-9790 was different for L-selectin and CD23 (83 v 6 μmol/L, respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester, CD23 was not lost under these conditions. The data show that extracellular ATP induces shedding of L-selectin and CD23 from B-CLL lymphocytes by an action mediated by the P2Z/P2X7 receptor. However, different membrane metalloproteases seem to mediate the shedding of L-selectin and CD23. © 1998 by The American Society of Hematology.
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Jørgensen, T. D., J. Gromada, K. Tritsaris, B. Nauntofte, and S. Dissing. "Activation of P2z purinoceptors diminishes the muscarinic cholinergic-induced release of inositol 1,4,5-trisphosphate and stored calcium in rat parotid acini. ATP as a co-transmitter in the stimulus-secretion coupling." Biochemical Journal 312, no. 2 (December 1, 1995): 457–64. http://dx.doi.org/10.1042/bj3120457.
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The effect of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and inositol phosphate production following stimulation with the muscarinic cholinergic agonist acetylcholine (ACh) was investigated in isolated rat parotid acinar cells. Stimulation of rat parotid acinar cells with ATP4- results in a rise in [Ca2+]i that is due to influx of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Stimulation with purinergic agonists revealed that both influx as well as Ca2+ release from intracellular stores was mediated through activation of P2z receptors. The Ca2+ mobilization from intracellular stores was due to production of Ins(1,4,5)P3 and was inhibited by U73122, an inhibitor of phospholipase C-coupled processes. Under Ca(2+)-free conditions ATP4- caused a dose-dependent inhibition (IC50 = 8 microM) of the ACh-evoked Ca2+ release. The inhibitory effect of ATP4- is due to activation of the P2z purinoceptors, which results in a strong reduction in the ACh-induced inositol phosphate production. Prestimulation with 100 microM ATP4- reduced the amount of Ins(1,4,5)P3 formed after maximal ACh stimulation by 91%. In conclusion, the inhibitory effect of ATP4- on the ACh-mediated response is due to interactions of the activated P2z receptor with the phospholipase C-coupled processes underlying the muscarinic cholinergic response.
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Coutinho-Silva, Robson, Jean-Luc Perfettini, Pedro M. Persechini, Alice Dautry-Varsat, and David M. Ojcius. "Modulation of P2Z/P2X7receptor activity in macrophages infected withChlamydia psittaci." American Journal of Physiology-Cell Physiology 280, no. 1 (January 1, 2001): C81—C89. http://dx.doi.org/10.1152/ajpcell.2001.280.1.c81.
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Given the role that extracellular ATP (ATPo)-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATPohas any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATPo-induced killing via P2Z/P2X7purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATPo, because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X7purinergic receptors. Incubation with ATPobut not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATPoeffects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATPo-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATPoto permeabilize macrophages to small molecules and by abrogating a sustained Ca2+influx previously associated with ATPo-induced apoptosis.
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Baricordi, OR, D. Ferrari, L. Melchiorri, P. Chiozzi, S. Hanau, E. Chiari, M. Rubini, and F. Di Virgilio. "An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes." Blood 87, no. 2 (January 15, 1996): 682–90. http://dx.doi.org/10.1182/blood.v87.2.682.bloodjournal872682.
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We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation.
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Fukushi, Yasue. "Heterologous desensitization of muscarinic receptors by P2Z purinoceptors in rat parotid acinar cells." European Journal of Pharmacology 364, no. 1 (January 1999): 55–64. http://dx.doi.org/10.1016/s0014-2999(98)00824-3.
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Coutinho-Silva, Robson, Luiz Anastácio Alves, Antônio Carlos Campos de Carvalho, Wilson Savino, and Pedro Muanis Persechini. "Characterization of P2Z purinergic receptors on phagocytic cells of the thymic reticulum in culture." Biochimica et Biophysica Acta (BBA) - Biomembranes 1280, no. 2 (April 1996): 217–22. http://dx.doi.org/10.1016/0005-2736(95)00293-6.
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Lammas, D. A., C. Stober, C. J. Harvey, N. Kendrick, S. Panchalingam, and D. S. Kumararatne. "ATP-Induced Killing of Mycobacteria by Human Macrophages Is Mediated by Purinergic P2Z(P2X7) Receptors." Immunity 7, no. 3 (September 1997): 433–44. http://dx.doi.org/10.1016/s1074-7613(00)80364-7.
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Lachish, M., E. Alzola, N. Chaib, M. Metioui, K. Grosfils, E. Kabre, A. Moran, A. Marino, and J. P. Dehaye. "Study of nonspecific cation channel coupled to P2z purinergic receptors using an acid load technique." American Journal of Physiology-Cell Physiology 271, no. 6 (December 1, 1996): C1920—C1926. http://dx.doi.org/10.1152/ajpcell.1996.271.6.c1920.
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The intracellular pH (pHi) of rat submandibular cells was measured by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The cells recovered from ammonium (30 mM) prepulse to their resting pHi within 10 min. Ethylisopropylamiloride (EIPA), an inhibitor of the Na+/H+ exchanger, slows the rate of pHi recovery. ATP (1 mM), in the presence of EIPA, increases the rate of recovery 3.7-fold in the absence or presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The recovery was blocked by the addition of 5 mM Mg2+ or 10 microM Coomassie blue. The response was elicited by 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate but not by ADP, UTP, adenyl (beta-gamma-methylene)-diphosphonate, 2-methylthioadenosine 5'-triphosphate, or muscarinic or beta-adrenergic agonists. The purinergic response was also observed when the cells were acidified by sodium propionate and could not be mimicked by the depolarization of the plasma membrane. Aluminum fluoride did not reproduce the response to ATP, suggesting that the observed response does not involve a high-molecular-weight GTP-binding protein. It is concluded that the activation of P2z receptors, probably by the opening of nonspecific cation channels, increases the permeability to protons in rat submandibular glands.
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Schilling, William P., William G. Sinkins, and Mark Estacion. "Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblasts." American Journal of Physiology-Cell Physiology 277, no. 4 (October 1, 1999): C755—C765. http://dx.doi.org/10.1152/ajpcell.1999.277.4.c755.
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Maitotoxin (MTX), a potent cytolytic agent, activates Ca2+ entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts was examined. MTX produced a time- and concentration-dependent increase in cytosolic free Ca2+ concentration that depended on extracellular Ca2+ and was relatively insensitive to blockade by extracellular lanthanides. MTX also produced a time- and concentration-dependent increase in plasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da), 2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately, 5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation, N-methyl-d-glucamine (167 Da). Thus MTX initially activates Ca2+-permeable cation channels and later induces the formation of large pores. These effects of MTX on plasmalemmal permeability are similar to those seen on activation of P2Z/P2X7 receptors in a variety of cell types, raising the intriguing possibility that MTX and P2Z/P2X7 receptor stimulation activate a common cytolytic pore.
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Tenneti, L., and B. R. Talamo. "Modulation of extracellular ATP-induced Ca2+ responses: role of protein kinases." Biochemical Journal 295, no. 1 (October 1, 1993): 255–61. http://dx.doi.org/10.1042/bj2950255.
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Evidence for the modulation of the P2z-purinoceptor for extracellular ATP in dissociated rat parotid cells is presented in studies using compounds that inhibit protein kinases. Preincubation of acinar cells with the protein kinase catalytic-site inhibitors K-252a and staurosporine, as well as with the regulatory-domain inhibitor sphingosine, specifically potentiates the elevation in cytosolic Ca2+ concentration ([Ca2+]i) mediated by extracellular ATP, but has no effect on the [Ca2+]i elevation mediated by muscarinic receptors through phospholipase C activation. Phorbol dibutyrate (PDBu), which activates protein kinase C (PKC), has no modulatory effect on ATP-mediated [Ca2+]i elevation. Further, pretreatment with PDBu does not reverse or block the effects of K-252a or sphinogosine, arguing against the involvement of PKC. Other pharmacological manipulations indicate that neither calmodulin-dependent nor cyclic-AMP-dependent kinases are involved. Neither the peak intracellular Ca2+ mobilization nor the sustained Ca2+ entry in response to carbachol or to a Ca2+ ionophore (4-bromo-A23187) is altered by the kinase inhibitors that potentiate the [Ca2+]i response to ATP, indicating that effects on the ATP response are not due to non-specific permeability changes, nor to decreased Ca2+ removal from the cytosol. ATP-mediated influx of Mn2+ as well as ATP-induced membrane depolarization are potentiated in cells preincubated with K-252a, directly demonstrating that cation influx is enhanced through a P2z-specific route. These results show that P2z responses (or purinoceptors) can be modulated and suggest that phosphorylation events are involved.
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el-Moatassim, C., and G. R. Dubyak. "A novel pathway for the activation of phospholipase D by P2z purinergic receptors in BAC1.2F5 macrophages." Journal of Biological Chemistry 267, no. 33 (November 1992): 23664–73. http://dx.doi.org/10.1016/s0021-9258(18)35890-3.
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Kaiho, Hiromi, Junko Kimura, Isao Matsuoka, and Hironori Nakanishi. "Effects of Anions on ATP-Activated Nonselective Cation Current in NG108-15 Cells." Journal of Neurophysiology 77, no. 5 (May 1, 1997): 2717–22. http://dx.doi.org/10.1152/jn.1997.77.5.2717.
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Kaiho, Hiromi, Junko Kimura, Isao Matsuoka, and Hironori Nakanishi. Effects of anions on ATP-activated nonselective cation current in NG108-15 cells. J. Neurophysiol. 77: 2717–2722, 1997. Extracellular ATP induces a nonselective cation current and elevates intracellular Ca2+ concentration via P2Z receptors in NG108-15 cells. We found that the ATP-induced nonselective cation current became larger in methanesulfonic acid (MS−) than in Cl− external solution. We therefore examined the effects of various external anions on the ATP-induced cation current with the use of the whole cell patch-clamp technique. The concentration-response curves for ATP were obtained in different anionic external solutions. The maximum current density ( I max) and the concentration of agonist that gives 50% of maximum response (EC50) value of ATP were obtained by fitting the curves with the use of the Hill coefficient of 2. The apparent I max decreased in the order of aspartic acid (Asp−) > MS− > F− > Cl− > Br− ≥ I−. The apparent EC50 values for ATP were shifted to the right in the sequence of Asp− < F− < MS− < Br− < Cl− < I−. Thus both I max and EC50 values were affected by anions, indicating that anions are mixed-type inhibitors of the ATP-induced current. The shift of the EC50 values of ATP indicates that anions interfere with ATP binding to the receptor. External Cl− was a noncompetitive inhibitor with respect to external Na+, a major cation carrying the ATP-induced current. We conclude that extracellular anions inhibit the ATP-induced nonselective cation current at least partly by interfering with ATP binding to the P2Z receptor, which is associated with the nonselective cation channels.
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Punj, Vasu, Olga Zaborina, Neelam Dhiman, Kim Falzari, M. Bagdasarian, and A. M. Chakrabarty. "Phagocytic Cell Killing Mediated by Secreted Cytotoxic Factors of Vibrio cholerae." Infection and Immunity 68, no. 9 (September 1, 2000): 4930–37. http://dx.doi.org/10.1128/iai.68.9.4930-4937.2000.
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ABSTRACT Vibrio cholerae strain VB1 secretes a number of enzymes into the outside medium that utilize ATP as a substrate. Such enzymes are found in the outside medium during the mid-log phase of growth, when the optical density at 650 nm is about 0.4, and they demonstrate nucleoside diphosphate kinase (Ndk), 5′ nucleotidase, and adenylate kinase (Ak) activities. We report that the filtered growth medium ofV. cholerae, as well as the flowthrough fraction of a green Sepharose column during fractionation of the growth medium, had very little cytotoxicity by itself towards macrophages and mast cells but exhibited significant cytotoxicity in the presence of exogenous ATP. Such fractions, harboring 5′ nucleotidase, Ndk, and presumably other ATP-utilizing enzymes, demonstrated enhanced macrophage and mast cell death; periodate-oxidized-ATP (oATP)-treated macrophage and mast cells or such cells exposed to 0.1 mM Mg2+, where surface-associated P2Z receptors could not be activated, were not susceptible to subsequent ATP addition. Microscopic visualization of mast cells clearly demonstrated cell morphological changes such as swelling, vacuolization, and nuclear fragmentation following treatment with ATP and the growth medium of V. cholerae; however, these effects were suppressed if the mast cells were pretreated with oATP. These results strongly imply that the secreted ATP-utilizing enzymes of V. cholerae modulate the external ATP levels of the macrophage and mast cells, leading to their accelerated death, presumably through activation of P2Z receptors. Thus, development of inhibitors for such enzymes may reduce the level of V. cholerae infection; alternatively, mutations in such genes may eliminate V. cholerae survival in the gut and contribute to a safer live vaccine.
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Nuttle, L. C., and G. R. Dubyak. "Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes." Journal of Biological Chemistry 269, no. 19 (May 1994): 13988–96. http://dx.doi.org/10.1016/s0021-9258(17)36744-3.
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FORESTA, Carlo, Marco ROSSATO, Andrea NOGARA, Francesco GOTTARDELLO, Paola BORDON, and Francesco DI VIRGILIO. "Role of P2-purinergic receptors in rat Leydig cell steroidogenesis." Biochemical Journal 320, no. 2 (December 1, 1996): 499–504. http://dx.doi.org/10.1042/bj3200499.
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The present study investigated the effects of extracellular ATP on the intracellular calcium ion concentration ([Ca2+]i) and testosterone production in isolated adult rat Leydig cells. This nucleotide caused an increase in [Ca2+]i, with a maximal effect at a concentration of 100 µM ATP, comprising a rapid initial spike followed by a long-lasting plateau. The first rapid spike was dependent on the release of Ca2+ from internal stores, since it also occurred in Ca2+-free medium and was abolished after depletion of internal stores with thapsigargin. The second, long-lasting, phase was dependent on the influx of Ca2+ from the extracellular medium. After 3 h of incubation, extracellular ATP stimulated testosterone secretion in a dose-dependent manner, with a maximal effect at 100 µM. Activation of steroidogenesis by ATP was fully dependent on the presence of Ca2+ in the external medium. Among different nucleotides, only ATP, adenosine 5´-[γ-thio]triphosphate, UTP, benzoylbenzoic-ATP and 2-methylthio-ATP were effective in inducing both the rise in [Ca2+]i and testosterone secretion. These effects were blocked by preincubation of Leydig cells with oxidized ATP, an inhibitor of the P2Z-purinergic receptor subtype. These results show that rat Leydig cells possess P2-purinergic receptors whose activation triggers an increase in [Ca2+]i due to the release of Ca2+ from internal stores and Ca2+ influx from the external medium. The stimulatory effect of extracellular ATP on testosterone secretion seems to be coupled to the influx of Ca2+ from the external medium.
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Pacaud, P., R. Malam-Souley, G. Loirand, and C. Desgranges. "ATP raises [Ca2+]i via different P2-receptor subtypes in freshly isolated and cultured aortic myocytes." American Journal of Physiology-Heart and Circulatory Physiology 269, no. 1 (July 1, 1995): H30—H36. http://dx.doi.org/10.1152/ajpheart.1995.269.1.h30.
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In vascular smooth muscle, extracellular ATP induces an increase in intracellular [Ca2+] ([Ca2+]i). Various agonists have been used to characterize P2-purinoceptor subtypes involved in the ATP-induced [Ca2+]i rise, measured by indo 1 fluorescence, in both freshly isolated and cultured rat aortic smooth muscle cells. alpha, beta-Methylene-ATP increased [Ca2+]i via Ca2+ entry through P2x-receptor channels in freshly isolated but not in cultured cells. 2-Methylthio-ATP and ADP failed to release Ca2+ via P2y-receptor activation in freshly isolated cells, whereas such a response was obtained in cultured cells. UTP, by stimulating P2u receptors, released Ca2+ from intracellular stores in both freshly isolated and cultured cells. These results suggest that, in the course of the culture process, P2x-receptor activation-induced responses were lost, whereas P2y-receptor activation-induced [Ca2+]i rise appeared, these two phenomena being independent. Responses to P2x-receptor agonist were lost in all culture conditions, whereas functional P2y receptors appeared only in cells that were stimulated with serum to induce cell cycle progression. The phenotypic modulation of vascular myocytes was therefore associated with a change in the functional P2-purinoceptor subtypes.
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Zaborina, Olga, Xiaoming Li, Guofeng Cheng, Vinayak Kapatral, and A. M. Chakrabarty. "Secretion of ATP-utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?" Molecular Microbiology 31, no. 5 (March 1999): 1333–43. http://dx.doi.org/10.1046/j.1365-2958.1999.01240.x.
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Firestein, B. L., M. Xing, R. J. Hughes, C. U. Corvera, and P. A. Insel. "Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells." American Journal of Physiology-Renal Physiology 271, no. 3 (September 1, 1996): F610—F618. http://dx.doi.org/10.1152/ajprenal.1996.271.3.f610.
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We have characterized the signaling pathways of purinergic receptors present on the renal epithelial cell line, Madin-Darby canine kidney (MDCK, D1 subclone). Several lines of evidence are consistent with the conclusion that coexisting P2u and P2y receptors release arachidonic acid and metabolites (AA) from MDCK-D1 cells: 1) relative potencies of nucleotide analogues, 2) blockade of P2y agonist- but not P2u agonist-mediated release by suramin, and 3) additivity by 2-methylthio-ATP and UTP. Differences exist between the signaling pathways of the two receptors: pertussis toxin treatment partially inhibits P2u- but not P2y-mediated AA release, and P2y (but not P2u) receptors appear to stimulate D-myo-inositol 1,4,5-trisphosphate production. P2u-receptor occupancy results in both homologous and heterologous desensitization; P2y-receptor occupancy elicits only homologous desensitization. Both receptors stimulate phosphatidylcholine hydrolysis via phospholipase C activation. However, AA release appears to result from phospholipid deacylation by phospholipase A2 activation, rather than from alternate pathways that may include PLC activation. These results demonstrate for the first time that two subtypes of P2-purinergic receptors, P2u and P2y receptors, coexist on a single renal epithelium cell type and that these two receptor subtypes can promote AA release, probably via activation of PLA2.
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KUNAPULI, Satya P., and James L. DANIEL. "P2 receptor subtypes in the cardiovascular system." Biochemical Journal 336, no. 3 (December 15, 1998): 513–23. http://dx.doi.org/10.1042/bj3360513.
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Extracellular nucleotides have been implicated in a number of physiological functions. Nucleotides act on cell-surface receptors known as P2 receptors, of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. Furthermore, nucleotides are also released from damaged or broken cells. Thus during vascular injury nucleotides play an important role in haemostasis through activation of platelets, modulation of vascular tone, recruitment of neutrophils and monocytes to the site of injury, and facilitation of adhesion of leucocytes to the endothelium. Nucleotides also moderate these functions by generating nitric oxide and prostaglandin I2 through activation of endothelial cells, and by activating different receptor subtypes on vascular smooth muscle cells. In the heart, P2 receptors regulate contractility through modulation of L-type Ca2+ channels, although the molecular mechanisms involved are still under investigation. Classical pharmacological studies have identified several P2 receptor subtypes in the cardiovascular system. Molecular pharmacological studies have clarified the nature of some of these receptors, but have complicated the picture with others. In platelets, the classical P2T receptor has now been resolved into three P2 receptor subtypes: the P2Y1, P2X1 and P2TAC receptors (the last of these, which is coupled to the inhibition of adenylate cyclase, is yet to be cloned). In peripheral blood leucocytes, endothelial cells, vascular smooth muscle cells and cardiomyocytes, the effects of classical P2X, P2Y and P2U receptors have been found to be mediated by more than one P2 receptor subtype. However, the exact functions of these multiple receptor subtypes remain to be understood, as P2-receptor-selective agonists and antagonists are still under development.
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el-Moatassim, C., and G. R. Dubyak. "Dissociation of the pore-forming and phospholipase D activities stimulated via P2z purinergic receptors in BAC1.2F5 macrophages. Product inhibition of phospholipase D enzyme activity." Journal of Biological Chemistry 268, no. 21 (July 1993): 15571–78. http://dx.doi.org/10.1016/s0021-9258(18)82295-5.
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Verspohl, E. J., B. Johannwille, A. Waheed, and H. Neye. "Effect of purinergic agonists and antagonists on insulin secretion from INS-1 cells (insulinoma cell line) and rat pancreatic islets." Canadian Journal of Physiology and Pharmacology 80, no. 6 (June 1, 2002): 562–68. http://dx.doi.org/10.1139/y02-079.
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The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 [Formula: see text] 0.0032 μM) and inhibiting insulin release (EC50 [Formula: see text] 0.32 μM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPβS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 [Formula: see text] 0.02 μM) and inhibitory (EC50 [Formula: see text] 0.32 μM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 [Formula: see text] 15 μM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 [Formula: see text] 2 μM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 μM) antagonized the insulinotropic effect of ATP (0.01 μM) and UTP (0.32 μM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 μM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPβS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 μM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 μM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effective via P2Y receptors and the PLC-system and not via P2X receptors; it inhibits insulin release at high concentrations by being metabolized to adenosine.Key words: purinergic receptors, insulin release, ATP.
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Spranzi, E., JY Djeu, SL Hoffman, PK Epling-Burnette, and DK Blanchard. "Lysis of human monocytic leukemia cells by extracellular adenosine triphosphate: mechanism and characterization of the adenosine triphosphate receptor." Blood 82, no. 5 (September 1, 1993): 1578–85. http://dx.doi.org/10.1182/blood.v82.5.1578.1578.
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Abstract The present study shows that extracellular adenosine triphosphate (ATP) has the capacity to mediate dose-dependent lysis of the monocytic leukemia cell line THP-1. The lysis, assessed by 51Cr release, was found to be selective for ATP, because adenosine diphosphate (ADP) or other nucleotides were less effective in their ability to lyse the cells. The amount of 51Cr released was particularly enhanced by the stimulation of the cells with 1,000 U/mL of interferon gamma (IFN- gamma) for 3 days, and the sensitivity was time and dose dependent. Analysis of the mechanism of lysis indicated that the fully ionized form, ATP4-, mediated the lysis, because the addition of cation chelators or the absence of the divalent cations, Ca2+ and Mg2+, in the culture medium of a 6-hour 51Cr release assay increased the percent specific lysis. Therefore, the ATP receptors on THP-1 cells were classified as P2Z purinoceptors. Moreover, it is shown here that the Ca2+/calmodulin complex plays a role in the regulation of the lysis by extracellular ATP of THP-1 cells, because antagonists of this complex, such as trifluoperazine or KN-62, were found to inhibit the ATP- mediated cell lysis.
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Spranzi, E., JY Djeu, SL Hoffman, PK Epling-Burnette, and DK Blanchard. "Lysis of human monocytic leukemia cells by extracellular adenosine triphosphate: mechanism and characterization of the adenosine triphosphate receptor." Blood 82, no. 5 (September 1, 1993): 1578–85. http://dx.doi.org/10.1182/blood.v82.5.1578.bloodjournal8251578.
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The present study shows that extracellular adenosine triphosphate (ATP) has the capacity to mediate dose-dependent lysis of the monocytic leukemia cell line THP-1. The lysis, assessed by 51Cr release, was found to be selective for ATP, because adenosine diphosphate (ADP) or other nucleotides were less effective in their ability to lyse the cells. The amount of 51Cr released was particularly enhanced by the stimulation of the cells with 1,000 U/mL of interferon gamma (IFN- gamma) for 3 days, and the sensitivity was time and dose dependent. Analysis of the mechanism of lysis indicated that the fully ionized form, ATP4-, mediated the lysis, because the addition of cation chelators or the absence of the divalent cations, Ca2+ and Mg2+, in the culture medium of a 6-hour 51Cr release assay increased the percent specific lysis. Therefore, the ATP receptors on THP-1 cells were classified as P2Z purinoceptors. Moreover, it is shown here that the Ca2+/calmodulin complex plays a role in the regulation of the lysis by extracellular ATP of THP-1 cells, because antagonists of this complex, such as trifluoperazine or KN-62, were found to inhibit the ATP- mediated cell lysis.
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Klaver, Dominik, and Martin Thurnher. "Control of Macrophage Inflammation by P2Y Purinergic Receptors." Cells 10, no. 5 (May 4, 2021): 1098. http://dx.doi.org/10.3390/cells10051098.
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Macrophages comprise a phenotypically and functionally diverse group of hematopoietic cells. Versatile macrophage subsets engage to ensure maintenance of tissue integrity. To perform tissue stress surveillance, macrophages express many different stress-sensing receptors, including purinergic P2X and P2Y receptors that respond to extracellular nucleotides and their sugar derivatives. Activation of G protein-coupled P2Y receptors can be both pro- and anti-inflammatory. Current examples include the observation that P2Y14 receptor promotes STAT1-mediated inflammation in pro-inflammatory M1 macrophages as well as the demonstration that P2Y11 receptor suppresses the secretion of tumor necrosis factor (TNF)-α and concomitantly promotes the release of soluble TNF receptors from anti-inflammatory M2 macrophages. Here, we review macrophage regulation by P2Y purinergic receptors, both in physiological and disease-associated inflammation. Therapeutic targeting of anti-inflammatory P2Y receptor signaling is desirable to attenuate excessive inflammation in infectious diseases such as COVID-19. Conversely, anti-inflammatory P2Y receptor signaling must be suppressed during cancer therapy to preserve its efficacy.
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McCoy, David E., Amanda L. Taylor, Brian A. Kudlow, Katherine Karlson, Margaret J. Slattery, Lisa M. Schwiebert, Erik M. Schwiebert, and Bruce A. Stanton. "Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors." American Journal of Physiology-Renal Physiology 277, no. 4 (October 1, 1999): F552—F559. http://dx.doi.org/10.1152/ajprenal.1999.277.4.f552.
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Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na+ absorption [measured via Na+ short-circuit current[Formula: see text])] and stimulated Cl− secretion [measured via Cl−short-circuit current ([Formula: see text])]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate [Formula: see text] and[Formula: see text]. By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that [Formula: see text] is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas[Formula: see text] is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit[Formula: see text] across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate [Formula: see text]through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.
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Sauzeau, Vincent, Hélène le Jeune, Chrystelle Cario-Toumaniantz, Nathalie Vaillant, Alain-Pierre Gadeau, Claude Desgranges, Elizabeth Scalbert, Pierre Chardin, Pierre Pacaud, and Gervaise Loirand. "P2Y1, P2Y2, P2Y4, and P2Y6 receptors are coupled to Rho and Rho kinase activation in vascular myocytes." American Journal of Physiology-Heart and Circulatory Physiology 278, no. 6 (June 1, 2000): H1751—H1761. http://dx.doi.org/10.1152/ajpheart.2000.278.6.h1751.
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In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca2+ sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y1, P2Y2, P2Y4, or P2Y6 receptor subtypes. Our data demonstrate that P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.
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Ruan, Huai-Zhen, Lori A. Birder, William C. de Groat, Changfeng Tai, James Roppolo, Charles A. Buffington, and Geoffrey Burnstock. "Localization of P2X and P2Y Receptors in Dorsal Root Ganglia of the Cat." Journal of Histochemistry & Cytochemistry 53, no. 10 (June 27, 2005): 1273–82. http://dx.doi.org/10.1369/jhc.4a6556.2005.
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The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X5 receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y2 = P2Y4>P2X3>P2X2 = P2X7>P2X6>P2X1 = P2X4>P2Y1. P2X3, P2Y2, and P2Y4 receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y2, P2X1, P2X3, P2X4, and P2X6 receptor staining was detected in small- and medium-diameter neurons. However, P2Y4, P2X2, and P2X7 staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X3 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y4 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y2 receptor-positive neurons also stained for IB4, CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X3-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y1 receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.
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Hurley, T. W., D. D. Shoemaker, and M. P. Ryan. "Extracellular ATP prevents the release of stored Ca2+ by autonomic agonists in rat submandibular gland acini." American Journal of Physiology-Cell Physiology 265, no. 6 (December 1, 1993): C1472—C1478. http://dx.doi.org/10.1152/ajpcell.1993.265.6.c1472.
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In dose-dependent fashion, extracellular ATP reduces the increase in cytosolic Ca2+ concentration ([Ca2+]o) due to mobilization of cellular Ca2+ stores by both epinephrine [half-maximal inhibitory concentration (IC50) = 35.7 +/- 12.9 microM; Hill coefficient (NH) = -2.0 +/- 0.7, n = 8] and by carbachol (IC50 = 27.0 +/- 7.0 microM, NH = -2.3 +/- 0.7, n = 9). Inhibition is due to ATP4- but does not result from any emptying or inaccessibility of Ca2+ stores, which are readily mobilized by thapsigargin in the presence of ATP4-. Reduction of Ca2+ mobilization is rapid but is not due to direct interference by ATP with the interaction of carbachol or epinephrine with their respective cell surface receptors. A benzoyl derivative of ATP, 3'-O-(4-benzoyl) adenosine 5'-triphosphate (BZATP) is more potent than ATP in reducing [Ca2+]i due to mobilization of stored Ca2+ by either carbachol or epinephrine (IC50 for carbachol = 3.9 +/- 0.4 microM, NH = -3.2 +/- 0.5; IC50 for epinephrine = 3.8 +/- 0.2, NH = -2.6 +/- 0.7, n = 3) but GTP, UTP, ADP, and adenosine do not inhibit mobilization of stored Ca2+ by either carbachol or epinephrine. Neither ATP nor BZATP prevents the influx of extracellular Ca2+ stimulated by carbachol or epinephrine. These results suggest that ATP inhibits Ca2+ mobilization by autonomic neurotransmitters after occupation of P2Z purinoceptors.
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Kennedy, Charles. "That was then, this is now: the development of our knowledge and understanding of P2 receptor subtypes." Purinergic Signalling 17, no. 1 (February 1, 2021): 9–23. http://dx.doi.org/10.1007/s11302-021-09763-0.
Full textAbstract:
AbstractP2 receptors are present in virtually all tissues and cell types in the human body, and they mediate the physiological and pharmacological actions of extracellular purine and pyrimidine nucleotides. They were first characterised and named by Geoff Burnstock in 1978, then subdivided into P2X and P2Y purinoceptors in 1985 on the basis of pharmacological criteria in functional studies on native receptors. Molecular cloning of receptors in the 1990s revealed P2X receptors to comprise seven different subunits that interact to produce functional homo- and heterotrimeric ligand-gated cation channels. A family of eight P2Y G protein–coupled receptors were also cloned, which can form homo- and heterodimers. Deep insight into the molecular mechanisms of agonist and antagonist action has been provided by more recent determination of the tertiary and quaternary structures of several P2X and P2Y receptor subtypes. Agonists and antagonists that are highly selective for individual subtypes are now available and some are in clinical use. This has all come about because of the intelligence, insight and drive of the force of nature that was Geoff Burnstock.
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Turner, Clare M., Brian F. King, Kaila S. Srai, and Robert J. Unwin. "Antagonism of endogenous putative P2Y receptors reduces the growth of MDCK-derived cysts cultured in vitro." American Journal of Physiology-Renal Physiology 292, no. 1 (January 2007): F15—F25. http://dx.doi.org/10.1152/ajprenal.00103.2006.
Full textAbstract:
P2Y receptors couple to G proteins and either mobilize intracellular Ca2+ or alter cAMP levels to modulate the activity of Ca2+- and cAMP-sensitive ion channels. We hypothesize that increased ion transport into the lumen of MDCK cysts can osmotically drive fluid movement and increase cyst size. Furthermore, activation of the adenylate cyclase/cAMP pathway may trigger cell proliferation via an extracellular signal-related kinase cascade. To test this hypothesis, several P2Y receptor inhibitors were used on the MDCK in vitro model of renal cyst formation. The nonspecific P2 receptor inhibitors reactive blue 2 and suramin reduced cyst growth significantly, as did PPADS and, to a lesser extent, the P2Y1-specific antagonist MRS2179. Cyst growth was reduced by ∼50% when ATP was removed from the culture medium with apyrase, although stable analogs of ATP failed to increase cyst size. The nonselective P2X receptor inhibitor Coomassie brilliant blue G was ineffective at reducing cyst growth, suggesting no involvement of P2X receptors. Finally, the presence of selective inhibitors of ERK activation (either PD98059 or U0126) greatly reduced cyst growth, whereas in untreated cysts ERK activity was observed to increase with time. We conclude that stimulation of endogenous P2Y receptors by extracellular ATP increases growth of MDCK cysts via cAMP-dependent activation of the ERK pathway. P2Y receptor antagonists may have therapeutic potential in reducing cyst size and slowing disease progression; although further studies in vitro and in vivo are needed to investigate the specificity and role of these P2Y receptors in renal cystic diseases.
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Xia, Shen-Ling, Lanjun Wang, Melanie N. Cash, Xueling Teng, Ruth A. Schwalbe, and Charles S. Wingo. "Extracellular ATP-induced calcium signaling in mIMCD-3 cells requires both P2X and P2Y purinoceptors." American Journal of Physiology-Renal Physiology 287, no. 2 (August 2004): F204—F214. http://dx.doi.org/10.1152/ajprenal.00281.2003.
Full textAbstract:
Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular Ca2+signaling and downstream cellular function. The purpose of this study was to determine whether P2 receptors were present in mIMCD-3 cells, a mouse inner medullary collecting duct cell line, and if so, to examine their link with intracellular Ca2+homeostasis. To monitor intracellular Ca2+concentration ([Ca2+]i), experiments were conducted using the fluorescent dye fura 2. ATP (0.1–100 μM) produced a dose-dependent increase in [Ca2+]iin a physiological Ca2+-containing solution, with an EC50of 2.5 μM. The P2-receptor antagonist PPADS reduced the effect of ATP on [Ca2+]i, and the P1-receptor agonist adenosine caused only a small increase in [Ca2+]i. Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [Ca2+]i, indicating P2Y receptors were involved in this process. In a Ca2+-free bath solution, thapsigargin and ATP induced intracellular Ca2+release from an identical pool. Nucleotides caused an increase in [Ca2+]iin the potency order of UTP = ATP > ATPγS > ADP > UDP that is best fitted with the P2Y2subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca2+]ias did ATP, a small but sustained increase in [Ca2+]ioccurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca2+influx. The sustained increase in [Ca2+]icould be blocked by either nonselective cation channel blockers Gd3+or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd3+or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca2+]iwas significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X1and P2Y2receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular Ca2+signaling.
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Lommen, Julian, Julika Detken, Katharina Harr, Charlotte von von Gall, and Amira A. H. Ali. "Analysis of Spatial and Temporal Distribution of Purinergic P2 Receptors in the Mouse Hippocampus." International Journal of Molecular Sciences 22, no. 15 (July 28, 2021): 8078. http://dx.doi.org/10.3390/ijms22158078.
Full textAbstract:
ATP and other nucleotides are important glio-/neurotransmitters in the central nervous system. They bind to purinergic P2X and P2Y receptors that are ubiquitously expressed in various brain regions modulating various physiological and pathophysiological processes. P2X receptors are ligand-gated ion channels mediating excitatory postsynaptic responses whereas P2Y receptors are G protein-coupled receptors mediating slow synaptic transmission. A variety of P2X and P2Y subtypes with distinct neuroanatomical localization provide the basis for a high diversity in their function. There is increasing evidence that P2 receptor signaling plays a prominent role in learning and memory and thus, in hippocampal neuronal plasticity. Learning and memory are time-of-day-dependent. Moreover, extracellular ATP shows a diurnal rhythm in rodents. However, it is not known whether P2 receptors have a temporal variation in the hippocampus. This study provides a detailed systematic analysis on spatial and temporal distribution of P2 in the mouse hippocampus. We found distinct spatial and temporal distribution patterns of the P2 receptors in different hippocampal layers. The temporal distribution of P2 receptors can be segregated into two large time domains, the early to mid-day and the mid to late night. This study provides an important basis for understanding dynamic P2 purinergic signaling in the hippocampal glia/neuronal network.
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Ecke, Denise, Theodor Hanck, Mohan E. Tulapurkar, Rainer Schäfer, Matthias Kassack, Rolf Stricker, and Georg Reiser. "Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor." Biochemical Journal 409, no. 1 (December 11, 2007): 107–16. http://dx.doi.org/10.1042/bj20070671.
Full textAbstract:
Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP–P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.
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Ren, Yong, Xiaoju Zou, Li Fang, and Qing Lin. "Involvement of Peripheral Purinoceptors in Sympathetic Modulation of Capsaicin-Induced Sensitization of Primary Afferent Fibers." Journal of Neurophysiology 96, no. 5 (November 2006): 2207–16. http://dx.doi.org/10.1152/jn.00502.2006.
Full textAbstract:
Purinoceptors are distributed in primary afferent terminals, where transmission of nociceptive information is modulated by these receptors. In the present study, we evaluated whether the activation or blockade of purinoceptors of subtypes P2X and P2Y in the periphery affected the sensitization of primary afferents induced by intradermal injection of capsaicin (CAP) and examined their role in sympathetic modulation of sensitization of primary nociceptive afferents. Afferent activity was recorded from single Aδ- and C-primary afferent fibers in the tibial nerve in anesthetized rats. Peripheral pretreatment with α,β-methylene adenosine 5′-triphosphate (α,β-meATP), a P2X-selective receptor agonist, could potentiate the CAP-induced enhancement of responses of Aδ- and C-primary afferent nociceptive fibers to mechanical stimuli in sympathetically intact rats. After sympathetic denervation, the enhanced responses of both Aδ- and C-fibers after CAP injection were dramatically reduced. However, this reduction could be restored when P2X receptors were activated by α,β-meATP. A blockade of P2X receptors by pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid could significantly reduce the CAP-induced sensitization of Aδ- and C-fibers. Pretreatment with uridine 5′-triphosphate, a P2Y-selective receptor agonist, did not significantly affect or restore the CAP-induced sensitization of Aδ- and C-fibers under sympathetically intact or sympathectomized conditions. Our study supports the view that ATP plays a role in modulation of primary afferent nociceptor sensitivity mainly by P2X receptors. Combined with our previous study, our data also provide further evidence that the sensitization of primary afferent nociceptors is subject to sympathetic modulation by activation of P2X as well as α1-adrenergic receptors.
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Mitrovic, Sandra, Houchaima Ben-Tekaya, Eva Koegler, Jean Gruenberg, and Hans-Peter Hauri. "The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi." Molecular Biology of the Cell 19, no. 5 (May 2008): 1976–90. http://dx.doi.org/10.1091/mbc.e07-10-0989.
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Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment.
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Miyagi, Yasushi, and John H. Zhang. "α,β-Methylene ATP enhances P2Y4 contraction of rabbit basilar artery." American Journal of Physiology-Heart and Circulatory Physiology 286, no. 4 (April 2004): H1546—H1551. http://dx.doi.org/10.1152/ajpheart.00926.2003.
Full textAbstract:
Interactions between different selective P2 receptor agonists have been used as tools to identify different P2 receptor subtypes. In the present study, we examined the P2 receptor subtypes and the mechanisms of potentiation of UTP contraction (P2Y contraction) by α,β-methylene ATP [(2-carboxypiperazin-4-yl)propyl-1-phosphanoic acid (CPP), a P2X agonist] using isometric tension in the denuded rabbit basilar artery. We made the following observations: 1) a predominant P2X receptor contraction was observed in the rabbit ear artery by the rank order of CPP » 2-methylthioATP > ATP > UTP; 2) functional P2Y receptors were observed in the rabbit basilar artery by the rank order of UTP » ATP = CPP = 2-methylthioATP; 3) CPP potentiated UTP-, ATP-, and ATPγS-induced contractions, possibly by activation of P2Y4 receptors because ATPγS does not activate P2Y6 receptors; and 4) ectonucleotidase did not play a predominant role in the potentiative effect of CPP because Evans blue, Ca2+-free medium, or divalent cation Ni2+ did not affect the effect of CPP. Evans blue potentiated the contraction by UTP but not by ATP or ATPγS. We conclude that CPP enhanced P2Y4-mediated contraction in the rabbit basilar artery, and the influence by ectonucleotidases on CPP-potentiation remains unclear.
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Inscho, Edward W., Anthony K. Cook, Vy Mui, and Jason Miller. "Direct assessment of renal microvascular responses to P2-purinoceptor agonists." American Journal of Physiology-Renal Physiology 274, no. 4 (April 1, 1998): F718—F727. http://dx.doi.org/10.1152/ajprenal.1998.274.4.f718.
Full textAbstract:
Studies were performed to determine the responsiveness of rat juxtamedullary afferent arterioles to receptor-selective P2-purinoceptor agonists. Experiments were performed in vitro using the blood perfused juxtamedullary nephron technique, combined with videomicroscopy. Renal perfusion pressure was set at 110 mmHg and held constant. Basal afferent arteriolar diameter averaged 22.0 ± 0.6 μm ( n = 69). Stimulation with 0.1, 1.0, 10, and 100 μM ATP ( n = 10) elicited a concentration-dependent vasoconstriction averaging 8 ± 2, 17 ± 2, 21 ± 4, and 23 ± 5%, respectively. A nearly identical afferent arteriolar vasoconstriction was observed in response to the P2X-selective agonist β,γ-methylene ATP ( n = 10); however, another P2X agonist, α,β-methylene ATP, evoked marked receptor desensitization ( n = 10). Vessel diameter decreased by ∼7 ± 2, 16 ± 2, 23 ± 3, and 22 ± 3%, respectively, over the same concentration range. The P2Y-selective agonist, 2-methylthio-ATP, evoked only a modest vasoconstriction, whereas UTP and adenosine 5′- O-(3-thiotriphosphate) (ATPγS) reduced afferent diameter markedly at concentrations >1.0 μM. Afferent arteriolar diameter decreased by 5 ± 4, 31 ± 8, and 72 ± 8% during UTP administration ( n = 7) at concentrations of 1.0, 10, and 100 μM, respectively. Similarly, ATPγS ( n = 6) decreased afferent diameter by 16 ± 2, 58 ± 8, and 98 ± 3%, respectively, over the same concentration range. Nitric oxide synthesis inhibition with N ω-nitro-l-arginine did not significantly alter the afferent arteriolar response to ATP but did potentiate ATP-mediated arcuate artery vasoconstriction. The following data suggest the presence of multiple P2 receptors on juxtamedullary afferent arterioles and are consistent with classification of those receptors as members of the P2X- and P2Y2 (P2U)-receptor subtypes.
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Marques, Rita D., Pauline I. A. de Bruijn, Mads V. Sorensen, Markus Bleich, Helle A. Praetorius, and Jens Leipziger. "Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL)." American Journal of Physiology-Renal Physiology 302, no. 4 (February 15, 2012): F487—F494. http://dx.doi.org/10.1152/ajprenal.00570.2011.
Full textAbstract:
Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (mTAL) including luminal and basolateral P2Y2 receptors (Jensen ME, Odgaard E, Christensen MH, Praetorius HA, Leipziger J. J Am Soc Nephrol 18: 2062–2070, 2007). In addition, we found evidence for a basolateral P2X receptor. Here, we investigate the effect of basolateral ATP on NaCl absorption in isolated, perfused mouse mTALs using the electrical measurement of equivalent short-circuit current ( I′sc). Nonstimulated mTALs transported at a rate of 1,197 ± 104 μA/cm2 ( n = 10), which was completely blockable with luminal furosemide (100 μM). Basolateral ATP (100 μM) acutely (1 min) and reversibly reduced the absorptive I′sc. After 2 min, the reduction amounted to 24.4 ± 4.0% ( n = 10). The nonselective P2 receptor antagonist suramin blocked the effect. P2Y receptors were found not to be involved in this effect. The P2X receptor agonist 2-methylthio ATP mimicked the ATP effect, and the P2X receptor antagonist periodate-oxidized ATP blocked it. In P2X7−/− mice, the ATP effect remained unaltered. In contrast, in P2X4−/− mice the ATP-induced inhibition of transport was reduced. A comprehensive molecular search identified P2X4, P2X5, and P2X1 receptor subunit mRNA in isolated mouse mTALs. These data define that basolateral ATP exerts a significant inhibition of Na+ absorption in mouse mTAL. Pharmacological, molecular, and knockout mouse data identify a role for the P2X4 receptor. We suggest that other P2X subunits like P2X5 are part of the P2X receptor complex. These data provide the novel perspective that an ionotropic receptor and thus a nonselective cation channel causes transport inhibition in an intact renal epithelium.