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Coexpression of Ligand-gated P2X and G Protein-coupled P2Y Receptors in Smooth Muscle

1998; Elsevier BV; Volume: 273; Issue: 8 Linguagem: Inglês

10.1074/jbc.273.8.4695

ISSN

1083-351X

Autores

Karnam S. Murthy, Gabriel M. Makhlouf,

Tópico(s)

Neurological Complications and Syndromes

Resumo

P2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca2+ release, and contraction that were abolished by U-73122 and guanosine 5′-O-(3-thio)diphosphate, and partly inhibited (50–60%) by pertussis toxin (PTX). ATP analogs (adenosine 5′-(α,β-methylene)triphosphate, adenosine 5′-(β,γ-methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca2+ influx and contraction that were abolished by nifedipine and in Ca2+-free medium. Micromolar concentrations of ATP stimulated both Ca2+ influx and Ca2+ release.ATP and UTP activated Gq/11 and Gi3 in gastric and aortic smooth muscle and heart membranes, Gq/11 and Gi1 and/or Gi2 in liver membranes, and Go and Gi1–3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by Gαq/11-dependent activation of phospholipase (PL) C-β1 and Gβγi3-dependent activation of PLC-β3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to Gαq/11, Gβ, PLC-β1, or PLC-β3, and completely inhibited by the following combinations (PLC-β1 and PLC-β3 antibodies; Gαq/11 and Gβ antibodies; PLC-β1 and Gβ antibodies; PTX with either PLC-β1 or Gαq/11 antibody).The pattern of responses implied that P2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-β1 via Gαq/11 and to PLC-β3 via Gβγi3. These receptors co-exist with ligand-gated P2X1 receptors activated by ATP analogs and high levels of ATP. P2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca2+ release, and contraction that were abolished by U-73122 and guanosine 5′-O-(3-thio)diphosphate, and partly inhibited (50–60%) by pertussis toxin (PTX). ATP analogs (adenosine 5′-(α,β-methylene)triphosphate, adenosine 5′-(β,γ-methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca2+ influx and contraction that were abolished by nifedipine and in Ca2+-free medium. Micromolar concentrations of ATP stimulated both Ca2+ influx and Ca2+ release. ATP and UTP activated Gq/11 and Gi3 in gastric and aortic smooth muscle and heart membranes, Gq/11 and Gi1 and/or Gi2 in liver membranes, and Go and Gi1–3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by Gαq/11-dependent activation of phospholipase (PL) C-β1 and Gβγi3-dependent activation of PLC-β3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to Gαq/11, Gβ, PLC-β1, or PLC-β3, and completely inhibited by the following combinations (PLC-β1 and PLC-β3 antibodies; Gαq/11 and Gβ antibodies; PLC-β1 and Gβ antibodies; PTX with either PLC-β1 or Gαq/11 antibody). The pattern of responses implied that P2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-β1 via Gαq/11 and to PLC-β3 via Gβγi3. These receptors co-exist with ligand-gated P2X1 receptors activated by ATP analogs and high levels of ATP. P2 receptors have been classified recently into two classes comprising ligand-gated cationic channels or P2X receptors and G protein-coupled P2Yreceptors (1Fredholm B.B. Abbracchio M.P. Burnstock G. Daly J.W. Harden T.K. Jacobson K.A. Leff P. Williams M. Pharmacol. Rev. 1994; 46: 143-156PubMed Google Scholar, 2Fredholm B.B. Abbracchio M.P. Burnstock G. Dubyak G.R. Harden T.K. Jacobson K.A. Schwabe U. Williams M. Trends Pharmacol. Sci. 1997; 18: 79-82Abstract Full Text PDF PubMed Scopus (322) Google Scholar); P2U and P2T receptors have been subsumed into the P2Y class of receptors. The term P2 recognizes the fact that purine and pyrimidine nucleotides can act as preferential ligands of various receptor subtypes (2Fredholm B.B. Abbracchio M.P. Burnstock G. Dubyak G.R. Harden T.K. Jacobson K.A. Schwabe U. Williams M. Trends Pharmacol. Sci. 1997; 18: 79-82Abstract Full Text PDF PubMed Scopus (322) Google Scholar). Up to seven P2X receptor subtypes (3Valera S. Hussy N. Evans R.J. Adami N. North R.A. Surprenant A. Buell G. Nature. 1994; 371: 516-519Crossref PubMed Scopus (901) Google Scholar, 4Brake A.J. Wagenbach M.J. Julius D. Nature. 1994; 371: 519-523Crossref PubMed Scopus (843) Google Scholar, 5Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (894) Google Scholar, 6Chen C. Akopian A.N. Sivilotti L. Colquhoun D. Burnstock G. Wood J.N. Nature. 1995; 377: 428-430Crossref PubMed Scopus (919) Google Scholar, 7Bo X. Zhang Y. Nassar M. Burnstock G. Schoepfer R. FEBS Lett. 1995; 375: 129-133Crossref PubMed Scopus (258) Google Scholar, 8Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 9Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1507) Google Scholar) and eight P2Y receptor subtypes (10Webb T.E. Simon J. Krishek B.J. Bateson A.N. Smart T.G. King B.J. Burnstock G. Barnard E.A. FEBS Lett. 1993; 324: 219-225Crossref PubMed Scopus (468) Google Scholar, 11Lustig K.D. Shiau A.K. Brake A.J. Julius D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5113-5117Crossref PubMed Scopus (532) Google Scholar, 12Communi D. Pirotton S. Parmentier M. Boeynaems J.M. J. Biol. Chem. 1995; 270: 30849-30852Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 13Schachter J.B. Li Q. Boyer J.L. Nicholas R.A. Harden T.K. Br. J. Pharmacol. 1996; 118: 167-173Crossref PubMed Scopus (223) Google Scholar, 14Webb T.E. Henderson D. King B.F. Wang S. Simon J. Bateson A.N. Burnstock G. Barnard E.A. Mol. Pharmacol. 1996; 50: 258-265PubMed Google Scholar, 15Chang K. Hanaoka K. Kumada M. Takuwa Y. J. Biol. Chem. 1995; 270: 26152-26158Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 16Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) have been cloned from mammalian and avian species. Fuller understanding of the functions subserved by discrete receptor subtypes is hampered by the organization of native P2X receptors into homopolymers or heteropolymers (5Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (894) Google Scholar) and by the co-existence of P2X and P2Yreceptors on the same cell (17Pacaud P. Malam-Souley R. Loirand G. Desgranges C. Am. J. Physiol. 1995; 269: H30-H36PubMed Google Scholar). Earlier classifications based on agonist potency profiles had been confounded by the paucity of selective antagonists and radioligands (2Fredholm B.B. Abbracchio M.P. Burnstock G. Dubyak G.R. Harden T.K. Jacobson K.A. Schwabe U. Williams M. Trends Pharmacol. Sci. 1997; 18: 79-82Abstract Full Text PDF PubMed Scopus (322) Google Scholar), and by the rapid degradation of some nucleotides, mainly ATP and 2-methylthio-ATP, by ecto-nucleotidases (18Crack B.E. Buekers M.W. McKechnie K.C.W. IJzerman A.P. Leff P. Br. J. Pharmacol. 1994; 113: 1432-1438Crossref PubMed Scopus (84) Google Scholar), and the interconversion of adenine and uridine nucleotides by ecto-nucleoside diphosphokinases (19Nicholas R.A. Watt W.C. Lazarowski E.R. Li Q. Harden T.K. Mol. Pharmacol. 1996; 50: 224-229PubMed Google Scholar, 20Harden T.K. Lazarowski E.R. Boucher R.C. Trends Pharmacol. Sci. 1997; 18: 43-46Abstract Full Text PDF PubMed Google Scholar). P2X1 is the main P2X receptor subtype expressed in visceral and vascular smooth muscle (21Vulchanova L. Arvidsson U. Riedl M. Wang J. Buell G. Surprenant A. North R.A. Elde R. Proc. Natl. Acad. Aci. U. S. A. 1996; 93: 8063-8067Crossref PubMed Scopus (367) Google Scholar), whereas P2X2and P2X3 are the main receptor subtypes expressed in peripheral sensory ganglia (8Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 21Vulchanova L. Arvidsson U. Riedl M. Wang J. Buell G. Surprenant A. North R.A. Elde R. Proc. Natl. Acad. Aci. U. S. A. 1996; 93: 8063-8067Crossref PubMed Scopus (367) Google Scholar, 22Buell G. Collo G. Rassendren F. Eur. J. Neurosci. 1996; 8: 2221-2228Crossref PubMed Scopus (241) Google Scholar, 23Surprenant A. Buell G. North R.A. Trends Neurosci. 1995; 18: 224-229Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Both P2X1 and P2X3 receptors have high affinity for ATP and AMP-PCP 1The abbreviations used are: AMP-PCP, adenosine 5′-(α,β-methylene)triphosphate; AMP-CPP, adenosine 5′-(β,γ-methylene)triphosphate; CHAPS, 3-[(cholamidopropyl)dimethylammonio]-1-propane sulfonic acid; IP3, inositol 1,4,5-trisphosphate; GDPβS, guanosine 5′-O-(3-thio)diphosphate; DPCPX, cyclopentyl-1,3-dipropylxanthine; CGS- 15943, 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine; PTX,pertussis toxin; GTPγS, guanosine 5′-O-(3-thio)triphosphate; PLC, phospholipase C; [Ca2+]i, intracellular Ca2+; PI, phosphoinositol. and are rapidly desensitized (23Surprenant A. Buell G. North R.A. Trends Neurosci. 1995; 18: 224-229Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 24Werner P. Seward E.P. Buell G.N. North R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15485-15490Crossref PubMed Scopus (129) Google Scholar). P2X2, P2X4, and P2X6 receptors are the predominant receptor subtypes expressed in the adult brain where they are present in various heteromeric combinations; these receptor subtypes exhibit lower affinity for ATP, are insensitive to AMP-PCP, and are not readily desensitized (7Bo X. Zhang Y. Nassar M. Burnstock G. Schoepfer R. FEBS Lett. 1995; 375: 129-133Crossref PubMed Scopus (258) Google Scholar, 8Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 22Buell G. Collo G. Rassendren F. Eur. J. Neurosci. 1996; 8: 2221-2228Crossref PubMed Scopus (241) Google Scholar, 23Surprenant A. Buell G. North R.A. Trends Neurosci. 1995; 18: 224-229Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Their insensitivity to AMP-PCP restricts the usefulness of this analog as a radioligand for all but the P2X1 and P2X3 receptor subtypes (24Werner P. Seward E.P. Buell G.N. North R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15485-15490Crossref PubMed Scopus (129) Google Scholar). P2Y receptors exhibit variable affinity for purine and pyrimidine nucleotides. P2Y1 are purinoceptors and are adenine nucleotide-specific (10Webb T.E. Simon J. Krishek B.J. Bateson A.N. Smart T.G. King B.J. Burnstock G. Barnard E.A. FEBS Lett. 1993; 324: 219-225Crossref PubMed Scopus (468) Google Scholar, 13Schachter J.B. Li Q. Boyer J.L. Nicholas R.A. Harden T.K. Br. J. Pharmacol. 1996; 118: 167-173Crossref PubMed Scopus (223) Google Scholar), whereas P2Y2receptors (P2U in earlier classifications) have equal affinity for adenine and uridine nucleotide triphosphates (UTP ≥ ATP) (11Lustig K.D. Shiau A.K. Brake A.J. Julius D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5113-5117Crossref PubMed Scopus (532) Google Scholar, 19Nicholas R.A. Watt W.C. Lazarowski E.R. Li Q. Harden T.K. Mol. Pharmacol. 1996; 50: 224-229PubMed Google Scholar). P2Y3, P2Y4, and P2Y6are pyrimidinoceptors: P2Y4 is UTP selective whereas P2Y3 and P2Y6 are UDP selective (14Webb T.E. Henderson D. King B.F. Wang S. Simon J. Bateson A.N. Burnstock G. Barnard E.A. Mol. Pharmacol. 1996; 50: 258-265PubMed Google Scholar, 19Nicholas R.A. Watt W.C. Lazarowski E.R. Li Q. Harden T.K. Mol. Pharmacol. 1996; 50: 224-229PubMed Google Scholar). The functional status of P2Y5 which has low homology to other P2Y receptors has not been resolved (16Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 25Webb T.E. Kaplan M.G. Barnard E.A. Biochem. Biophys. Res. Commun. 1996; 219: 105-110Crossref PubMed Scopus (99) Google Scholar), while the P2Y7 receptor has now been identified as the leukotriene B4 receptor (26Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu A. Nature. 1997; 387: 620-624Crossref PubMed Scopus (853) Google Scholar). P2Y receptors are variously coupled to pertussis toxin-sensitive and -insensitive G proteins which activate or inhibit various effector enzymes including phospholipase C-β (PLC-β) (15Chang K. Hanaoka K. Kumada M. Takuwa Y. J. Biol. Chem. 1995; 270: 26152-26158Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 16Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 27Cowen D.S. Sanders M. Dubyak G.R. Biochim. Biophys. Acta. 1990; 1053: 195-203Crossref PubMed Scopus (31) Google Scholar, 28Dubyak G.R. El-Motassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar, 29Lazarowski E.R. Harden T.K. J. Biol. Chem. 1994; 269: 11830-11836Abstract Full Text PDF PubMed Google Scholar, 30Harden T.K. Boyer J.S. Nicholas R.A. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 541-579Crossref PubMed Google Scholar), phospholipase D (31Irving H.R. Exton J.H. J. Biol. Chem. 1987; 262: 3440-3443Abstract Full Text PDF PubMed Google Scholar, 32Xie M.S. Dubyak G.R. Biochem. J. 1991; 278: 81-89Crossref PubMed Scopus (41) Google Scholar), phospholipase A2 (33Lazarowski E.R. Boucher R.C. Harden T.K. Am. J. Physiol. 1994; 266: C406-C415Crossref PubMed Google Scholar), and adenylyl cyclase (28Dubyak G.R. El-Motassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar, 30Harden T.K. Boyer J.S. Nicholas R.A. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 541-579Crossref PubMed Google Scholar,34Post S.R. Jacobson J.P. Insel P.A. J. Biol. Chem. 1996; 271: 2029-2032Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). ATP, UTP, and AMP-PCP can mobilize Ca2+ and elicit contractile responses in vascular and visceral smooth muscle suggesting that both P2X and P2Y receptors are present (15Chang K. Hanaoka K. Kumada M. Takuwa Y. J. Biol. Chem. 1995; 270: 26152-26158Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 17Pacaud P. Malam-Souley R. Loirand G. Desgranges C. Am. J. Physiol. 1995; 269: H30-H36PubMed Google Scholar, 35Gerwins P. Fredholm B.B. J. Biol. Chem. 1992; 267: 16081-16087Abstract Full Text PDF PubMed Google Scholar, 36Trezise D.J. Michel A.D. Grahames C.B.A. Khakh B.S. Surprenant A. Humphrey P.P.A. Naunyn-Schmiedeberg's Arch. Pharmacol. 1995; 351: 603-609Crossref PubMed Scopus (42) Google Scholar, 37Najbar A.T. Li C.G. Rand M.J. Br. J. Pharmacol. 1996; 118: 537-542Crossref PubMed Scopus (8) Google Scholar). Their co-existence raises the question as to which receptor subtype mediates preferentially the action of the endogenous ligand, ATP. In the present study, we have used a series of purine and pyrimdine agonists to characterize P2 receptors in dispersed gastric smooth muscle cells and identify the signaling pathways to which they are coupled. Comparative studies characterized the coupling of P2Y receptors to G proteins in vascular smooth muscle, heart, liver, and brain. P2X1 and P2Y2 receptors were shown to co-exist on gastric smooth muscle cells and to mediate Ca2+ mobilization and muscle contraction via three distinct pathways. UTP and nanomolar concentrations of ATP activated exclusively P2Y2 receptors, whereas micromolar concentrations of ATP activated additionally P2X1 receptors. The pattern suggests that contraction induced by purine and pyrimidine nucleotides may be preferentially mediated by G protein-coupled receptors. Smooth muscle cells were isolated from the circular muscle layer of rabbit stomach by sequential enzymatic digestion, filtration, and centrifugation as described previously (38Bitar K.N. Makhlouf G.M. Nature. 1982; 297: 72-74Crossref PubMed Scopus (61) Google Scholar, 39Bitar K.N. Makhlouf G.M. Science. 1982; 216: 531-533Crossref PubMed Scopus (115) Google Scholar, 40Murthy K.S. Zhang K-M. Jin J-G. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 265: G660-G671PubMed Google Scholar). The cells were resuspended in enzyme-free medium consisting of 120 mm NaCl, 4 mm KCl, 2.6 mm KH2PO4, 2 mm CaCl2, 0.6 mmMgCl2, 25 mm HEPES, 14 mm glucose, and 2.1% Eagle's essential amino acid mixture. The muscle cells were harvested by filtration through 500-μm Nitex mesh and centrifuged twice at 350 × g for 10 min. In some experiments, the muscle cells were reversibly permeabilized using the Trans.Port reagent (Life Technologies, Inc.) as described previously (40Murthy K.S. Zhang K-M. Jin J-G. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 265: G660-G671PubMed Google Scholar). The cells were washed in Ca2+- and Mg2+-free HEPES medium and re-suspended in a medium containing 10 mm NaCl, 140 mm KCl, 2.4 mm MgCl2, and 10 mm HEPES. Trans.Port reagent (15 μl/ml) was added with or without GDPβS (10 μm) and the mixture incubated at 31 °C for 20 min. Permeabilization was terminated by addition of Stop solution (30 μl/ml) and the cell suspension centrifuged for 15 min at 350 ×g. The cells were resuspended in control HEPES medium containing 0.1% bovine serum albumin and incubated at 31 °C for 1 h. The resealed cells were shown to exclude trypan blue and respond to contractile agonists and depolarizing concentrations of KCl (20 mm) but not to 2 mm CaCl2 or inositol 1,4,5-trisphosphate (1 μm) (40Murthy K.S. Zhang K-M. Jin J-G. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 265: G660-G671PubMed Google Scholar). The effectiveness of GDPβS was tested by measuring its ability to abolish the contractile response to the contractile agonist, cholecystokinin octapeptide (40Murthy K.S. Zhang K-M. Jin J-G. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 265: G660-G671PubMed Google Scholar). The expression of G proteins and PLC-β isozymes was determined by Western blot analysis as described previously (41Murthy K.S. Coy D.C. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 42Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1996; 50: 870-877PubMed Google Scholar, 43Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1995; 47: 1172-1179PubMed Google Scholar). Homogenates prepared from dispersed gastric muscle cells were solubilized on ice for 1 h in 20 mm Tris (pH 8.0), 1 mm dithiothreitol, 100 mm NaCl, and 0.5% sodium cholate. The suspension was centrifuged at 13,000 × g for 5 min. Solubilized proteins were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. The blots were incubated for 12 h at 4 °C with subtype-specific G protein or PLC-β antibodies, and then for 1 h with secondary antibody conjugated with horseradish peroxidase. The bands were identified by enhanced chemiluminescence. A technique of selective receptor protection previously used to determine the co-existence and function of various G protein-coupled receptors (44Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1992; 263: G269-G276PubMed Google Scholar, 45Kuemmerle J.F. Martin D.C. Murthy K.S. Kellum J.M. Grider J.R. Makhlouf G.M. Mol. Pharmacol. 1992; 42: 1090-1096PubMed Google Scholar, 46Morini G. Kuemmerle J.F. Impicciatorre M. Grider J.R. Makhlouf G.M. J. Pharmacol. Exp. Ther. 1993; 264: 598-603PubMed Google Scholar, 47Murthy K.S. McHenry L. Makhlouf G.M. J. Pharmacol. Exp. Ther. 1995; 274: 300-306PubMed Google Scholar, 48Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1997; 272: 21317-21324Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was used to determine the presence and function of P2 receptor subtypes. The technique involves protection of one receptor subtype with selective agonists or antagonists followed by inactivation of all unprotected receptors with a low concentration ofN-ethylmaleimide (5 μm). Freshly dispersed muscle cells were incubated with one agonist (AMP-PCP, AMP-CPP, UTP, or ATP) at 31 °C for 2 min followed by addition of 5 μm N-ethylmaleimide for 20 min. The cells were centrifuged twice at 150 × g for 10 min and resuspended in control HEPES medium for 60 min to ensure complete re-sensitization. The contractile response of cells treated in this fashion was compared with the response of untreated cells. As previously shown (44Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1992; 263: G269-G276PubMed Google Scholar, 45Kuemmerle J.F. Martin D.C. Murthy K.S. Kellum J.M. Grider J.R. Makhlouf G.M. Mol. Pharmacol. 1992; 42: 1090-1096PubMed Google Scholar, 46Morini G. Kuemmerle J.F. Impicciatorre M. Grider J.R. Makhlouf G.M. J. Pharmacol. Exp. Ther. 1993; 264: 598-603PubMed Google Scholar, 47Murthy K.S. McHenry L. Makhlouf G.M. J. Pharmacol. Exp. Ther. 1995; 274: 300-306PubMed Google Scholar, 48Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1997; 272: 21317-21324Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), muscle cells incubated with N-ethylmaleimide without protective agent did not contract in response to receptor-linked agonists, but they responded fully upon addition of agents that bypass receptors (e.g. ionomycin, KCl, and forskolin), implying that post-receptor mechanisms were intact. Contraction of dispersed muscle cells was measured by scanning micrometry as described previously (38Bitar K.N. Makhlouf G.M. Nature. 1982; 297: 72-74Crossref PubMed Scopus (61) Google Scholar, 39Bitar K.N. Makhlouf G.M. Science. 1982; 216: 531-533Crossref PubMed Scopus (115) Google Scholar, 40Murthy K.S. Zhang K-M. Jin J-G. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 265: G660-G671PubMed Google Scholar). The length of 50 muscle cells treated with one concentration of a contractile agent was measured by scanning micrometry and compared with the length of 50 untreated muscle cells. All measurements were done in the presence of adenosine A1 and A2 antagonists (1 μm DPCPX and 0.1 μm CGS-15943, respectively) (47Murthy K.S. McHenry L. Makhlouf G.M. J. Pharmacol. Exp. Ther. 1995; 274: 300-306PubMed Google Scholar). Time course measurements were done at intervals ranging from 5 s to 5 min. As with other agonists, peak contraction was measured at 30 s and the response used to construct concentration-response curves. Contraction was expressed as the mean decrease in cell length from control in micrometers or as the percent decrease in cell length (range of control cell length in various experiments 96 ± 4 to 103 ± 5 μm). Cytosolic free Ca2+([Ca2+]i) was measured by fluorescence in suspensions of muscle cells loaded with the fluorescent Ca2+ dye, fura 2, as described previously (40Murthy K.S. Zhang K-M. Jin J-G. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 265: G660-G671PubMed Google Scholar, 45Kuemmerle J.F. Martin D.C. Murthy K.S. Kellum J.M. Grider J.R. Makhlouf G.M. Mol. Pharmacol. 1992; 42: 1090-1096PubMed Google Scholar). Autofluorescence of unloaded cells was determined in each suspension and subtracted from the fluorescence of fura 2-loaded cells. Measurements were done in the presence of adenosine A1 and A2 antagonists. Ca2+ levels were calculated under basal conditions and upon addition of agonist from the ratios of observed, minimal and maximal fluorescence (49Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). IP3 was measured in dispersed muscle cells by a radioreceptor assay which utilizes 3H-labeledd-myo-IP3 and bovine brain microsomes as described previously (41Murthy K.S. Coy D.C. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 42Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1996; 50: 870-877PubMed Google Scholar). Agonists were added for 30 s in the presence of adenosine A1 and A2 antagonists to 1 ml of muscle cell suspension (106 cells/ml) and the reaction terminated with an equal volume of ice-cold 10% perchloric acid. The supernatant was extracted and IP3 content in the aqueous phase was measured. The results were expressed as picomoles of IP3/106cells. PLC-β activity was determined in plasma membranes by a modification of the method of Uhing et al. (50Uhing R.J. Prpic V. Jiang H. Exton J.H. J. Biol. Chem. 1986; 261: 2140-2146Abstract Full Text PDF PubMed Google Scholar) as described previously (43Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1995; 47: 1172-1179PubMed Google Scholar,51Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 269: C969-C978Crossref PubMed Google Scholar). The membranes were isolated from dispersed muscle cells labeled with myo-[3H]inositol. PLC-β assay was initiated by addition of 0.4 mg of membrane protein to 25 mm Tris-HCl (pH 7.5), 0.5 mm EGTA, 10 mm MgCl2, 300 nm free Ca2+, 1 μm GTPγS, 5 mmphosphocreatine, and 50 units/ml creatine phosphokinase in a total volume of 0.4 ml. After incubation at 31 °C for 60 s, the reaction was terminated with 0.6 ml of 25% trichloroacetic acid (w/v). The supernatant was extracted four times with 2 ml of diethyl ether and the amount of labeled inositol phosphates in the aqueous phase was counted. All measurements were done in the presence of adenosine A1 and A2 antagonists. PLC-β activity was expressed as counts/min/mg protein/min. G proteins selectively activated by P2 receptor agonists in muscle cell membranes were identified by the method of Okomoto et al. (52Okomoto T. Ikezu T. Murayama Y. Ogata E. Nishimoto I. FEBS Lett. 1992; 305: 125-128Crossref PubMed Scopus (40) Google Scholar) as described previously (41Murthy K.S. Coy D.C. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 42Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1996; 50: 870-877PubMed Google Scholar, 48Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1997; 272: 21317-21324Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Muscle cell homogenates were centrifuged at 27,000 × g for 15 min, and the crude membranes solubilized for 60 min at 4 °C in 20 mm HEPES medium (pH 7.4) containing 2 mm EDTA, 240 mm NaCl, and 1% CHAPS. The membranes were diluted 10-fold and incubated at 37 °C with 60 nm[35S]GTPγS in a medium containing 10 mmHEPES (pH 7.4), 100 μm EDTA, and 10 mmMgCl2. The reaction was stopped with 10 volumes of 100 mm Tris-HCl medium (pH 8.0) containing 10 mmMgCl2, 100 mm NaCl, and 20 μmGTP, and the mixture incubated for 2 h on ice in wells precoated with specific G protein antibodies. The wells were washed three times with phosphate buffer solution containing 0.05% Tween 20 and the radioactivity from each well was counted. Coating with G protein antibodies (1:1000) was done after the wells were first coated with anti-rabbit IgG (1:1000) for 2 h on ice. The measurements were done in the presence of adenosine A1 and A2antagonists. In separate experiments on rabbit aortic smooth muscle, heart, liver, and whole brain, membranes were obtained by homogenization of these tissues without prior cell isolation. The homogenates were treated as described above for gastric smooth muscle cells. Results were expressed as means ± S.E. of n separate experiments and evaluated statistically using Student's t test for paired or unpaired data. Concentration-response curves were analyzed using the P.fit 6.0 program. d-myo-Inositol 1,4,5-trisphosphate assay system,myo-[3H]inositol, and [35S]GTPγS were obtained from Amersham; fura 2-acetoxymethyl ester from Molecular Probes; U-73122 from Biomol, Plymouth Meeting, PA; polyclonal antibodies to PLC-β1, PLC-β2, PLC-β3, PLC-β4, and Gβ from Santa Cruz Biotechnology, Santa Cruz, CA; polyclonal antibodies to Gαi1, Gαo, Gαi1–2, Gαi3, Gαq/11, and Gαs, and peptide fragments against which antibodies to Gαq/11 (QLNLKEYNLV) and Gαi3 (KNNKECGLY) were raised from Calbiochem; Gαi2 from Chemicon, Temecula, CA; DPCPX, AMP-PCP, AMP-CPP, and 2-methylthio-ATP from Research Biochemicals, Natick, MA; CGS-15943 from Ciba-Geigy, Summit, NJ; and all other reagents from Sigma. Exposure of muscle cells to 1 μm UTP or ATP caused immediate contraction that was virtually linear during the first 10 s and attained a peak in 30 s followed by a decline to lower levels (Fig. 1 A). The biphasic time course was identical to that observed with other contractile agonists (38Bitar K.N. Makhlouf G.M. Nature. 1982; 297: 72-74Crossref PubMed Scopus (61) Google Scholar, 53Bitar K.N. Bradford P.G. Putney Jr., J.W. Makhlouf G.M. J. Biol. Chem. 1986; 261: 16591-16596Abstract Full Text PDF PubMed Google Scholar). The peak response at 30 s was used to construct concentration-response curves. Prolonged exposure of muscle cells to purine or pyrimidine agonists resulted in time-dependent desensitization that was more rapid with P2X receptor agonists (e.g. AMP-PCP) than with P2Y receptor agonists (e.g., UTP) (Fig. 1 B). With either type of agonist, however, there was minimal desensitization (<2% of control response) during the initial 30-s period when peak response was measured. UTP, ATP, and ATP analogs caused concentration-dependent contraction of dispersed smooth muscle cells yielding curves with EC50 values of 33 ± 9 and 34 ± 6 nmfor ATP and UTP, respectively, 78 ± 17 and 85 ± 20 nm for AMP-PCP and AMP-CPP, respectively, and 178 ± 23 nm for 2-methylthio-ATP (Fig. 2). Except for the response to 2-methylthio-ATP, maximal contraction induced by all agonist (29 ± 3 to 30 ± 2% decrease in cell length) was similar to that elicited by other contractile agonists, such as cholecystokinin octapeptide (30 ± 3%) or acetylcholine (31 ± 4%). ATP, UTP, AMP-PCP, and AMP-CPP increased cytosolic Ca2+([Ca2+]i) in dispersed smooth muscle cells by 1-fold at 10 nm and by 3-fold at 10 μm (T

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