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Identification of a Uridine Nucleotide-Selective G-Protein-Linked Receptor That Activates Phospholipasec*

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Identification of a Uridine Nucleotide-Selective G-Protein-Linked Receptor That Activates Phospholipasec* THEJOURNAL OF BIOLMXCAL CHEMISTRY Vol. 269, No. 16, Issue of April 22, pp. 11830-11836, 1994 0 1994 by The American Soeiety for Biochemistry and Molecular Biology, Inc. Printed in USA. Identification of a Uridine Nucleotide-selective G-protein-linked Receptor That Activates PhospholipaseC* (Received for publication, October 21, 1993, and in revised form, January 14, 1994) Eduardo R. Lazarowski and T. Kendall Harden$ From the Department of Pharmacology, Uniuersity of North Carolina, School of Medicine, Chapel Hill,North Carolina 27599 Incubation of C6-2B rat gliomacells withUDP or UTP Although care has been taken toprove that theP,,-purinergic resulted in atime- and concentration-dependent in- receptor that stimulates inositol lipid hydrolysis in man.y tar- crease in the accumulation of inositol phosphates. In get tissuesis a single receptor activated by both ATP and UTP contrast, ATP, ADP, and analogs of these nucleotides (8,9), this does not rule out thepossibility that separate recep- known to be effective agonists Pzv-, at Pm-, P,,-, P,,-, and tors for purines and pyrimidines occur. Indeed, studies meas- P,,-purinergic receptors all had no effect on inositol uring vascular smooth muscle responses (10-12) or metabolic phosphate levels inC6-2B cells. Pyrimidine nucleotides effects in liver (13) and in neutrophils or HL-60 cells (14, 15) stimulated inositol phosphate accumulation with an or-have led to the proposal that “pyrimidinergic” receptors exist der of potency of UDP > 5-BrUTP > UTP > dTDP > UDP (16).However, none of these havebeen unambiguously defined. glucose. values for UDP, 5-BrUTP,and UTP were 2.3 A large amount of information has accumulated on the physi- 0.5,9 3, and 57 10 respectively.A similar uridine * * m, ological release ofATP and ADP. However, outside of its storage nucleotide selectivity was observed for arachidonic acid release presumably occurring asa consequence ofacti- in high concentrations in platelet storage granules, little is vation of phospholipase 4. Cross-desensitization and known for UTP concerning its potential storage, release, and additivity experiments indicated thatUDP and UTP in- activity as an extracellular molecule. The presence of P,,-pu- teract with the same populationof receptors. The effect rinergic receptors that respond to extracellular UTP supports of uridine nucleotideson inositol phosphate accumula- the idea thatthis is an important extracellular signalingmol- tion was inhibited markedly by pretreatment of cells ecule, and potentially provides avenues for studying thephysi- with pertussis toxin.UDP also caused a guanine nucle- ological regulation of extracellular UTP levels. otide-dependent increase in inositol lipid hydrolysis in The inositol lipid signaling cascade is the major second mes- streptolysin-0-permeabilized cells. Takentogether senger response known to be associated with activation of P,- these results describe the existence of a novel uridine purinergic receptors (3). Both the P,,- and P,,-purinergic re- nucleotide receptor that is not activated by adenine ceptors activate phospholipase C in a broad range of tissues. nucleotides. This receptoris pharmacologically distinct However, this is not the only signaling response regulated by from the previously described P2”- and other P,-puriner-these receptors. For example, activation of a P,-purinergic re- gic receptors, and likely is a member of a new class of ceptor that appears to be distinct from the p,,-purinergic re- receptors for extracellular nucleotides. ceptor that activates phospholipase C results in a decrease in intracellular cyclic AMP accumulation in some target tissues (17-20). It is also likely that there are otherreceptors for ex- Extracellular release of adenine nucleotides from neurons, tracellular adenine and uridinenucleotides that have not been platelets, and other tissues results in activation of a broad identified, and we report here that C6-2B glioma cells express range of physiological responses (1, 2). Considerable progress a novel receptor that possesses a strict selectivity for uridine has been made in the last decade in the pharmacological de- nucleotides. Activation of this receptor results in activation of lineation of the cell surface receptors that are activated by phospholipase C through a guanine nucleotide-dependent per- extracellular ATP and ADP. These P,-purinergic receptors in- tussis toxin-sensitive pathway. The existence of a receptor that clude the P,,-, P,,-, P,,-, P,,-, and P,,-receptors, which are is activated by uridine nucleotides but not by adenine nucle- selectively activated by ATP, ADP, or various analogs of these otides adds support to the idea that extracellular UTP sub- two adenine nucleotides (1-3). The one exception to the adenine serves an important cell signaling function. nucleotide selectivity of these receptors is the so-called P,,- EXPERIMENTALPROCEDURES purinergic receptor, which is not only activated by ATP and ATPyS,’ but also is stimulatedby the pyrimidine, UTP (4-8). Cell Culture and Subcloning“C6-2B and C6 rat glioma cells and HT29 human colonic carcinoma cells were grownin Dulbecco’s modified Eagle’s medium supplemented with DMEM and5% fetal bovine serum * This work was supported by United States Public Health Service at 37 “C in a humidified atmosphere of 5% CO,, 95% air. To isolate Grants HL32322, GM38213,and GM29536. The costsof publication of homogeneous cell populations derived from single cloned cells, C6-2B this article were defrayed in part by the payment of page charges. This cells were seeded on 150-cm2 dishesat cell densities rangingfrom 10 to article must thereforebe hereby marked “aduertisement”in accordance 1000 cellddish. Attached isolated single cells were identifiedthe follow- with 18 U.S.C. Section 1734 solely to indicate this fact. ing day, and compact clusters (apparently clonal)of dividing cells were $ To whom correspondence should be addressed: Dept. of Pharmacol- isolated using 7-mm diameter cloning cylinders. The C6-2B cell clones ogy, UNC-CH School of Medicine, CB 7365, FLOB, Chapel Hill, NC were transferred to tissue culture flasks andnumbered in the order of 27599-7365. Tel.: 919-966-4816;Fax: 919-966-5640. The abbreviations used are: ATPyS, adenosine-5’-0-(3-thiotri- their isolation. Inositol Phosphate Assay-Confluent cells grown on 12-well plastic phosphate); a,p-MeATP,a$-methylene adenosine-5’-triphosphate; 5-BrUTP, 5-bromo-uridine-5‘-triphosphate;2-MeSATP, 2-methylthio- plates (Costar, Cambridge, MA) were incubated for 18 h in 0.5 ml of adenosine triphosphate; GTPyS, guanosine-5‘-0-(3-thiotriphosphate); HPLC, high perfomance liquid chromatography; Ins(1,4,5)P3, inositol 4,5)P4,inositol 1,3,4,5-tetrakisphosphate;DMEM, Dulbecco’s modified 1,4,5-trisphosphate; Ins(1,3,4)P3, inositol 1,3,4-trisphosphate;Ins(l,3, Eagle’s medium. 11830 This is an Open Access article under the CC BY license. Uridine Nucleotide Receptor 11831 inositol-free DMEM containing 10 pCi/ml [3Hlinositol.The cells were washed twice with HEPES-buffered (pH 7.4) DMEM (WDMEM) and preincubated with WDMEM containing 10 m~ LiCl for 15 min before challenge with agonists. In those experiments where desensitization was studied, the cells were preincubated with agonist for 20 min prior to addition of LiCl and rechallenged by agonist. Incubations were ter- 0 CONTROL minated by removal of the medium followed by addition of 5% trichlo- roacetic acid. The trichloroacetic acid extracts were neutralized by (3 x 2 ml) extractions with ethyl ether. Resolution of the individual inositol phosphates on Dowex AGl-X8 columns was performed as described previously (21). Quantitation of Individual Inositol Phosphates by HPLC-Samples were prepared as described above except that the cells were labeled with 100 pCi of [3Hlinositol in 0.5 ml of DMEM. Individual inositol phosphates were resolved using a Whatman Partisil 10 strong anion- exchange column (Alltech Applied Science, Deerfield, IL) and an am- monium phosphate gradient as described previously(22). Radioactivity was monitored on-linewith a Radiomatic Flo-one detector (Radiomatic I Instruments, Tampa, FL). 0 10 20 30 Arachidonic Acid Release-Confluent cells grown on 12-well plastic plates were labeled for6 hwith 0.5 pCiof [3Hlarachidonicacid in 0.5 ml TIME, min of DMEM. The cells were washed free of unincorporated radioactivity FIG. 1.Time course of inositol phosphate formation in C6-2B and challenged with agonists for 10 min. Released [3Hlarachidonicacid glioma cells. [3HlInositol-labeled C6-2B cells were challengedwith 300 was measured in the cell supernatant as reported previously (23). p~ carbachol (A),UTP (W), UDP (O),ATP (O),ATPyS (A), or vehicle( 0 ) Assay of Phospholipase CActivity in Permeabilized Cells -[3HlInosi- for the indicated times. Incubations were terminated by addition of 5% tol-prelabeled cells on 12-well plates were rinsed twice with phosphate- trichloroacetic acid and total inositol phosphates were collected and buffered saline and permeabilized by a 5-min incubation with 0.2 IU/ml measured as detailed under “Experimental Procedures.”The amount of streptolysin-0 inassay buffer containing 0.424 m~ CaCl,, 3 m~ MgCl,, [3Hlinositol phosphates present at t = 0 (-2000 cpm) was subtracted 2 m~ EGTA, 115 m~ KCl, 10 m~ HEPES, and 10 m~ LiCl (pH 7.4) as from the indicated values. Data represent the mean value of results described previously(8). Streptolysin-0 containing buffer was removed obtained with triplicate assays that are representative of results from and thecells wereincubated in 0.5 ml of assay
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