Dimerization of the Human Receptors for Prostacyclin and Thromboxane Facilitates Thromboxane Receptor-mediated cAMP Generation
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m405002200
ISSN1083-351X
AutoresStephen J. Wilson, Aoife M. Roche, Ekaterina Kostetskaia, Emer M. Smyth,
Tópico(s)Hormonal and reproductive studies
ResumoProstacyclin (PGI2) and thromboxane (TxA2) are biological opposites; PGI2, a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA2, a vasoconstrictor and platelet activator. The molecular mechanisms involved in the counterregulation of PGI2/TxA2 signaling are unclear. We examined the interaction of the receptors for PGI2 (IP) and TxA2 (TPα). IP-induced cAMP and TP-induced inositol phosphate generation were unaltered when the receptors were co-expressed in HEK 293 cells (IP/TPα-HEK). TP-cAMP generation, in response to TP agonists or a TP-dependent isoprostane, iPE2III, was evident in IP/TPα-HEK and in aortic smooth muscle cells, but not in cells expressing either receptor alone, or in IP-deficient aortic smooth muscle cells. Augmentation of TP-induced cAMP generation, with the IP agonist cicaprost, was ablated in IP-deficient cells and was independent of direct IP signaling. IP/TPα heterodimers were formed constitutively when the receptors were co-expressed, with no overt changes in ligand binding to the individual receptor sites. However, despite inefficient binding of iPE2III to either the IP or TPα, expressed alone or in combination, robust cAMP generation was evident in IP/TPα-HEK, suggesting the formation of an alternative receptor site. Thus, IP/TPα dimerization was coincident with TP-cAMP generation, promoting a "PGI2-like" cellular response to TP activation. This represents a previously unknown mechanism by which IP may limit the cellular effects of TP. Prostacyclin (PGI2) and thromboxane (TxA2) are biological opposites; PGI2, a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA2, a vasoconstrictor and platelet activator. The molecular mechanisms involved in the counterregulation of PGI2/TxA2 signaling are unclear. We examined the interaction of the receptors for PGI2 (IP) and TxA2 (TPα). IP-induced cAMP and TP-induced inositol phosphate generation were unaltered when the receptors were co-expressed in HEK 293 cells (IP/TPα-HEK). TP-cAMP generation, in response to TP agonists or a TP-dependent isoprostane, iPE2III, was evident in IP/TPα-HEK and in aortic smooth muscle cells, but not in cells expressing either receptor alone, or in IP-deficient aortic smooth muscle cells. Augmentation of TP-induced cAMP generation, with the IP agonist cicaprost, was ablated in IP-deficient cells and was independent of direct IP signaling. IP/TPα heterodimers were formed constitutively when the receptors were co-expressed, with no overt changes in ligand binding to the individual receptor sites. However, despite inefficient binding of iPE2III to either the IP or TPα, expressed alone or in combination, robust cAMP generation was evident in IP/TPα-HEK, suggesting the formation of an alternative receptor site. Thus, IP/TPα dimerization was coincident with TP-cAMP generation, promoting a "PGI2-like" cellular response to TP activation. This represents a previously unknown mechanism by which IP may limit the cellular effects of TP. PGI2 1The abbreviations used are: PGI2, prostacyclin; COX, cyclooxygenase; TxA2, thromboxane; SMC, smooth muscle cell; TP, receptor for TxA2; IP, receptor for PGI2; GPCR, G protein-coupled receptor; hIP, human IP; hTP, human TP; ASMC, aortic smooth muscle cell(s); hASMC, human ASMC; mASMC, mouse ASMC; WT, wild type; IPKO, IP knockout; PKA, protein kinase A; 8-Br-cAMP, 8-bromo-cyclic AMP.1The abbreviations used are: PGI2, prostacyclin; COX, cyclooxygenase; TxA2, thromboxane; SMC, smooth muscle cell; TP, receptor for TxA2; IP, receptor for PGI2; GPCR, G protein-coupled receptor; hIP, human IP; hTP, human TP; ASMC, aortic smooth muscle cell(s); hASMC, human ASMC; mASMC, mouse ASMC; WT, wild type; IPKO, IP knockout; PKA, protein kinase A; 8-Br-cAMP, 8-bromo-cyclic AMP. and TxA2 are the predominant products of cyclooxygenase (COX) metabolism of arachidonic acid formed in the macrovascular endothelium and platelets, respectively (1Vane J.R. Botting R.M. Am. J. 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Kvien T.K. Schnitzer T.J. N. Engl. J. Med. 2000; 343: 1520-1528Crossref PubMed Scopus (3700) Google Scholar). Recent work, using mice genetically deficient in the receptors for PGI2 (the IP) or TxA2 (the TP), demonstrated that the proliferative and platelet response to vascular injury was TP-mediated and was limited specifically by PGI2 (5Cheng Y. Austin S.C. Rocca B. Koller B.H. Coffman T.M. Grosser T. Lawson J.A. FitzGerald G.A. Science. 2002; 296: 539-541Crossref PubMed Scopus (719) Google Scholar). In addition, delivery of PGI2 synthase in vivo prevents proliferation and migration of SMC, key features of restenosis and atherosclerosis (10Harada M. Toki Y. Numaguchi Y. Osanai H. Ito T. Okumura K. Hayakawa T. Cardiovasc. Res. 1999; 43: 481-491Crossref PubMed Scopus (22) Google Scholar, 11Numaguchi Y. Naruse K. Harada M. Osanai H. Mokuno S. Murase K. Matsui H. Toki Y. Ito T. Okumura K. Hayakawa T. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 727-733Crossref PubMed Scopus (72) Google Scholar), whereas the antioxidant and antiplatelet actions of PGI2 delayed atherogenesis and may underlie the protection from cardiovascular disease afforded by female gender (12Egan K.M. Lowson J.A. Fries S. Koller B. Rader D.J. Smyth E.M. Fitzgerald G.A. Science. 2004; (in press)Google Scholar). Maintenance of the PGI2/TxA2 balance appears to be a critical regulator of vascular disease; however, the molecular mechanisms underlying the counterregulation of PGI2/TxA2 signaling have not been fully elucidated.A single gene encoding a G protein-coupled receptor (GPCR) has been reported for both mediators (13Nusing R.M. Hirata M. Kakizuka A. Eki T. Ozawa K. Narumiya S. J. Biol. Chem. 1993; 268: 25253-25259Abstract Full Text PDF PubMed Google Scholar, 14Ogawa Y. Tanaka I. Inoue M. Yoshitake Y. Isse N. Nakagawa O. Usui T. Itoh H. Yoshimasa T. Narumiya S. Nakao K. Genomics. 1995; 27: 142-148Crossref PubMed Scopus (35) Google Scholar), although in contrast to IP, where splice variants have not been described, two variants of TP, termed TPα and TPβ, have been identified (15Raychowdhury M.K. Yukawa M. Collins L.J. McGrail S.H. Kent K.C. Ware J.A. J. Biol. Chem. 1994; 269: 19256-19261Abstract Full Text PDF PubMed Google Scholar). IP is coupled to at least two signaling systems, namely the generation of intracellular cAMP and activation of PLC (16Narumiya S. FitzGerald G.A. J. Clin. Invest. 2001; 108: 25-30Crossref PubMed Scopus (462) Google Scholar). Both TPα and TPβ are coupled to PLC, whereas the former may activate and the latter may inhibit AC activity (16Narumiya S. FitzGerald G.A. J. Clin. Invest. 2001; 108: 25-30Crossref PubMed Scopus (462) Google Scholar). There is substantial evidence for reciprocal regulation between IP and TP. TPα, but not TPβ, is a target for IP-mediated, PKA-dependent phosphorylation, resulting in TPα desensitization (17Walsh M.T. Foley J.F. Kinsella B.T. J. Biol. Chem. 2000; 275: 20412-20423Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Similarly, U46619-mediated activation of TP enhances IP-mediated cAMP generation in human platelets (18Murray R. Shipp E. FitzGerald G.A. J. Biol. Chem. 1990; 265: 21670-21675Abstract Full Text PDF PubMed Google Scholar), where only TPα is expressed (19Habib A. FitzGerald G.A. Maclouf J. J. Biol. Chem. 1999; 274: 2645-2651Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar).The interaction between IP and TP may not, however, be limited to events occurring secondary to activation of their respective second messenger systems. GPCRs have long been considered to exist and function as independent monomeric units. However, GPCRs from both closely related and distinct subfamilies are capable of interacting physically with one another to form heterodimers (20Devi L.A. Trends Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 21Breitwieser G.E. Circ. Res. 2004; 94: 17-27Crossref PubMed Scopus (167) Google Scholar). Far from being a benign association, GPCR heterodimerization can substantially modify receptor function (20Devi L.A. Trends Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 21Breitwieser G.E. Circ. Res. 2004; 94: 17-27Crossref PubMed Scopus (167) Google Scholar). Signaling may change as a result of altered agonist affinity for the receptors, altered affinity of the receptors for their respective G proteins or signaling via alternate pathways (21Breitwieser G.E. Circ. Res. 2004; 94: 17-27Crossref PubMed Scopus (167) Google Scholar). Heterodimerization of the δ- and κ-opioid receptors, for example, creates a "new" receptor binding site that has a reduced affinity for individual δ- or κ-selective ligands but that can ligate cooperatively selective agonists to induce synergistic functional responses (22Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (963) Google Scholar).Heterodimers of the angiotensin II AT1 receptor and the bradykinin B2 receptor demonstrate increased activation of AT1-coupled G proteins in response to angiotensin II, a phenomenon that may underlie preeclamptic hypertension (23AbdAlla S. Lother H. el Massiery A. Quitterer U. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (403) Google Scholar). Thus, two opposing vascular mediators, angiotensin II, a vasoconstrictor, and bradykinin, a vasodilator, can alter the action of the other via a direct interaction of their receptors (23AbdAlla S. Lother H. el Massiery A. Quitterer U. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (403) Google Scholar). In the present study, we examined whether the interaction between two similarly opposing vascular mediators, PGI2 and TxA2, is also mediated via receptor interaction. We demonstrate that coexpression of IP and TPα, either endogenously or in an overexpression cell model, facilitated TP-mediated cAMP generation. The absence of the IP, in SMCs cultured from IP knockout (IPKO) mice or in HEK 293 cells, rendered the TP largely inactive with regard to AC activity. This interaction between IP and TPα is not dependent on IP-cAMP signaling, but is coincident with the formation of an IP/TPα heterodimer.EXPERIMENTAL PROCEDURESMaterials—Cyclic AMP radioimmunoassay kit, enhanced chemiluminescence kits, protein G-Sepharose, and all radiochemicals were purchased from Amersham Biosciences. Cell culture reagents, G418, and Albumax were obtained from Invitrogen. Complete protease inhibitor tablets were obtained from Roche Applied Science. IBOP, SQ 29548, iPE2III, and iPF2αIII were purchased from Cayman Chemical Co. (Ann Arbor, MI). H89 was obtained from Calbiochem. Isobutylmethylxanthine and deoxycholic acid were purchased from Sigma. Monoclonal anti-HA and anti-Myc were obtained from Covance (Richmond, CA). NuPAGE gels and buffers were purchased from Invitrogen. Secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA). Cicaprost was obtained from Schering AG under agreement.Epitope Tagging of hIP and hTP—The 9-amino acid hemagglutinin epitope (HA; YPYDVPDYA) or 10-amino acid Myc epitope (EQKLI-SEEDL) was inserted between the N-terminal initiator methionine and the second amino acid of the hTPα or hIP to generate HAhIP, MychIP, or HAhTPα. Generation of HAhIP was as described previously (24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To generate HAhTPα and MychIP, 5′-oligonucleotides that contained 3 miscellaneous bases, 6 bases encoding a HindIII site, the 3 miscellaneous bases immediately 5′ of the initiator methionine, 3 bases encoding a methionine, the epitope tag coding sequence, and 21 bases encoding amino acids 2–8 were generated. 3′-Oligonucleotides were complementary to the receptor coding sequence downstream of a unique restriction site (an EcoN47 site for hIP or a NotI site for hTPα). Using the hTPα or hIP cDNAs as templates, polymerase chain reactions were carried out to generate the 5′-HAhTP and 5′-MychIP fragments. The resulting products were cloned into PCR 2.1 (Qiagen, CA) and, following verification of the sequence, were excised using HindIII/NotI or HindIII/ EcoN47, as appropriate. Using the same enzymes, the 3′ fragment in pcDNA 3.1 (or pcDNA3.1 Hygro for MychIP) was generated, and the two receptor pieces were ligated to each other. The integrity of the splice site was verified by sequencing.Cell Culture and Transfection—HEK 293 cells (American Type Tissue Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, 25 mm HEPES, and 2 mm l-glutamine. Human aortic SMC (hAMSC, Biowhittaker Inc., Walkersville, MD) were cultured in smooth muscle cell basal medium supplemented with fetal bovine serum (5%), human recombinant epidermal growth factor (hEGF; 0.5 ng/ml), insulin (5 μg/ml), human recombinant fibroblast growth factor (hFGF; 2 ng/ml) plus gentamicin (50 μg/ml), and amphotericin-B (50 ng/ml). hASMC of passages 5–9 were used in experiments. Smooth muscle cells were isolated from WT or IPKO mouse aortic explants and grown in Dulbecco's modified Eagle's medium/F-12 Ham's medium supplemented with 20% heat-inactivated fetal bovine serum, 25 units/ml penicillin, 25 μg/ml streptomycin, and 2 mm l-glutamine. mASMC of passages 3–6 were used in experiments. All cells were maintained in a humidified atmosphere of 5% CO2, 95% air.For stable transfections, HEK 293 cells were seeded at 1.5 × 106 cells/100-mm dish and transfected the following day with 10 μgofDNA by liposome-mediated transfer (DOTAP), as described previously (25Smyth E.M. Li W.H. FitzGerald G.A. J. Biol. Chem. 1998; 273: 23258-23266Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 26Kallal L. Benovic J.L. Trends Pharmacol. Sci. 2000; 21: 175-180Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Stable transfectants were selected in the presence of G418 (0.5–1.5 mg/ml) and/or hygromycin (50–75 μg/ml).Western Blotting—Membranes or whole cell lysates were resolved (30 μg/lane) on NuPAGE (Invitrogen) 10% gels. HA-tagged or Myc-tagged receptors were visualized with anti-HA or anti-Myc (1:1000 dilution) in 5% nonfat milk in Tris-buffered saline (50 mm Tris, 250 mm NaCl, pH 7.6), containing 1% Tween 20 (TBS-T), for 1 h at room temperature followed by horseradish peroxidase-conjugated anti-mouse IgG (1:5000 dilution; Jackson Immunology), after first blocking with 5% nonfat milk in TBS-T for 2 h at room temperature. Antigen-antibody complexes were visualized by enhanced chemiluminescence.Membrane Preparation and Radioligand Binding—Membranes were prepared from confluent 100-mm dishes as described previously (24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Radioligand binding studies were carried out using membrane proteins (10 μg/reaction) in 10 mm HEPES, pH 7.4 (TP binding) containing 10 mm MnCl (IP binding). Radioligand displacement experiments were initiated by the addition of [3H]iloprost (4 nm) or [3H]SQ 29548 (40 nm), whereas saturation binding experiments utilized increasing concentrations of [3H]SQ 29548 (0–200 nm). Reactions were allowed to continue for 30 min at 30 °C and terminated by the addition of 3 ml of ice-cold wash buffer (10 mm HEPES, pH 7.4, 0.01% bovine serum albumin), followed by immediate filtration through GF/C filters that had been thoroughly soaked in the same ice-cold buffer. Following one wash with ice-cold wash buffer, radioactivity associated with the filters was quantified by scintillation counting. Nonspecific binding was measured in the presence of a 500-fold excess of unlabeled iloprost or SQ 29548, respectively. Saturation binding data were analyzed using GraphPad Prism 3.0 to calculate Kd and Bmax and to compare one- and two-site curve-fitting models (partial F-test).cAMP Measurements—Cells were grown to confluence in 12- or 6-well plates. hASMC and mASMC were pretreated overnight with 3 μm indomethacin to inhibit endogenous eicosanoid generation, followed by the addition of medium containing isobutylmethylxanthine (0.01 m) 30 min prior to agonist treatment. Cells were treated, and reactions were terminated by aspiration. cAMP was extracted with ice-cold 65% ethanol for 30 min. Samples were dried under vacuum and reconstituted in assay buffer, and cAMP was quantified by radioimmunoassay, as described previously (24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).Inositol Phosphate Production—Inositol phosphate production was assessed, as described previously (24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, cells were labeled overnight with 2 μCi/ml [3H]myoinositol. Thirty minutes prior to stimulation, cells were treated with 20 mm LiCl at 37 °C. After stimulation for 10 min at 37 °C, the reactions were terminated by aspiration. Total inositol phosphates were extracted with formic acid for 30 min at room temperature and neutralized using 5 m ammonia. Total inositol phosphates were recovered by anion exchange using Dowex 1-X8 AG anion exchange resin.Co-immunoprecipitation—All immunoprecipitation procedures were carried out at 4 °C. HEK 293 cells stably expressing hTPα or hIP or both were treated with 3 mm dithiobis(succinimidylpropionate) (Pierce) for 30 min and lysed in buffer A (150 mm NaCl, 1 mm EDTA, 20 mm Tris-HCl (pH 8.0), 10% glycerol, and a mixture of protease inhibitors) for 2 h at 4 °C. The resulting supernatants were precleared by a 1-h rotation with 100 μl of 10% (w/v) protein G-Sepharose to each tube. Anti-Myc- or anti-HA-protein G-Sepharose was prepared by adding 9 μg of anti-Myc ascites per lysate to 10% protein G-Sepharose followed by a 1-h rotation. MychIP or HAhTPα was immunoprecipitated from precleared lysates by adding 150 μl of anti-Myc- or anti-HA-protein G-Sepharose to each lysate and rotating for 16 h. Protein G was precipitated at 14,000 rpm for 1 min, washed three times with Buffer A, and resuspended in 10 μl of sample buffer (Nupage). Immunoblotting for HA or Myc was carried out as described above, using the appropriate biotinylated antibody (1:500) followed by peroxidase-labeled streptavidin.RESULTSGeneration of HEK 293 Cell Lines Expressing HA-hTPα or Coexpressing Both HA-hTPα and MychIP—We have described previously the generation of a HEK 293 cell line stably expressing HA-tagged hIP (hIP-HEK; see Ref. 24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Cell lines stably expressing HAhTPα (TPα-HEK), or coexpressing both MychIP and HAhTPα (IP/TPα-HEK), were generated in the present study in order to examine the effect of IP/TPα coexpression on TPα signaling.Lysates from each cell line were resolved by SDS-PAGE and immunoblotted with an anti-HA or anti-Myc antibody to establish that the receptors were being appropriately expressed. HAhTPα was observed as a broad complex of between 48 and 64 kDa in lysates derived from TPα-HEK or IP/TPα-HEK cells (Fig. 1). MychIP was observed as a 44–60-kDa complex in IP/TPα-HEK cell lysates (Fig. 1). This corresponds to the molecular weight of HAhIP previously observed in hIP-HEK cells (24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The broad molecular weight range of both hIP and hTPα is a result of receptor glycosylation (19Habib A. FitzGerald G.A. Maclouf J. J. Biol. Chem. 1999; 274: 2645-2651Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).Stimulation of hIP-HEK (see Ref. 24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) or IP/TPα-HEK with the prostacyclin analogue cicaprost for 5 min induced a concentration-dependent increase in intracellular cAMP (EC50 = 0.05 ± 0.02 nm, n = 4; Fig. 2) and inositol phosphate production (EC50 = 97.1 ± 33.3 nm, n = 4; Fig. 2), indicating that co-expression of hTPα did not alter hIP-mediated activation of two signaling systems when overexpressed in HEK 293 cells (24Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Stimulation of TPα-HEK with the specific TP-agonist U46619 resulted in increased inositol phosphate production (EC50 = 174.4 ± 65.2 nm, n = 3; Fig. 3), which was not altered significantly by co-expression of hIP (EC50 = 231.7 ± 40.8 nm, n = 3; Fig. 3). Much evidence supports ligation of the TP by isoprostanes, free radical-catalyzed products of arachidonic acid. Indeed, two TP-dependent isoprostanes (27Audoly L.P. Rocca B. Fabre J.E. Koller B.H. Thomas D. Loeb A.L. Coffman T.M. FitzGerald G.A. Circulation. 2000; 101: 2833-2840Crossref PubMed Scopus (183) Google Scholar), iPE2III (Fig. 3), and iPF2αIII (data not shown) stimulated inositol phosphate generation in TPα-HEK and IP/TPα-HEK, albeit with significantly higher EC50 values (3.56 ± 1.12 and 4.43 ± 0.63 μm, respectively) compared with U46619. Thus, similar to other studies, the addition of HA or Myc tags to the N terminus of IP and TP did not alter the expression or signal transduction properties of the receptor. Furthermore, co-expression of the receptors did not alter their discrete signal transduction properties.Fig. 2IP-mediated signaling in cells coexpressing MychIP and HAhTPα. IP/TPα-HEK were treated with increasing concentrations of cicaprost (5 min) and cAMP (closed circles) or inositol phosphates (open circles), quantified as indicated under "Experimental Procedures." Data are presented as mean -fold over basal ± S.E. from four experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3TP-mediated signaling in cells coexpressing MychIP and HAhTPα. TPα-HEK (A) or IP/TPα-HEK (B) were treated with U46619 (closed squares) or iPE2III (closed triangles) for 10 min, and inositol phosphates were quantified as described under "Experimental Procedures." Data are presented as mean -fold over basal ± S.E. from three or four experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TPα-mediated cAMP Formation—Treatment with the TP agonists IBOP or U46619 (100 nm, 10 min) induced a robust increase in cAMP levels in cells coexpressing HAhTPα and MychIP, but not in cells individually expressing the receptors or in mixed cultures of individually expressing cells (Fig. 4A). Pretreatment with the TP antagonist SQ 29548 partially reduced signaling by the TP agonists (Fig. 4B). Interestingly, iPE2III, but not iPF2αIII, also initiated an increase in cAMP in IP/TPα-HEK cells (Fig. 4A). In contrast to the TP agonists, the activity of iPE2III was insensitive to SQ 29548 (Fig. 4B), suggesting that this event was TP-independent. However, signaling was not observed in the absence of hTPα, demonstrating that inhibition with SQ 29548 is not sufficient to determine TP dependence (Figs. 4A and 5). Generation of cAMP in response to treatment of IP/TPα-HEK with IBOP, U46619, or iPE2III proved concentration-dependent, and only minor cAMP increments were observed at the highest concentration of TP agonist in cells expressing TPα alone (Fig. 5). These results indicate that cAMP formation in response to TP activation by TP agonists or iPE2III is dependent on the presence of both TPα and IP. We examined the biological relevance of this relationship in a cell model that endogenously expresses both IP and TP. cAMP production in response to IBOP or iPE2III was quantified in aortic smooth muscle cells isolated from humans or WT mice. Increased cAMP levels were observed in hASMC or WT mASMC following a 10-min treatment with IBOP or iPE2III (Fig. 6). SQ 29548 was partially effective against IBOP and ineffective against iPE2III (Fig. 6, E and F), in agreement with the HEK 293 cell data (Fig. 4B). IBOP or iPE2III treatment of ASMC isolated from IPKO mice resulted in minimal cAMP generation (Fig. 6, C and D). Thus, the absence of the IP in ASMC cultured from IPKO mice or in HEK 293 cells (Fig. 5) uncoupled the TP from activation of AC.Fig. 4Effect of coexpression of IP and TPα on TPα-induced cAMP generation. A, TPα-HEK (light gray bar), IP/TPα-HEK (open bar), or hIP-HEK cells (dark gray bar) or a mixed culture of TPα-HEK and IP-HEK cells (closed bar) were treated with a 100 nm concentration of the TP agonists U46619 and IBOP or the isoprostanes iPE2III and iPF2αIII for 10 min, and cAMP was quantified as indicated under "Experimental Procedures." Data are presented as the mean -fold over basal cAMP ± S.E. from 5–8 experiments each performed in duplicate. B, IP/TPα-HEK were pretreated with (open bar) or without (closed bar) SQ 29548 (1 μm, 30 min) prior to treatment with a 100 nm concentration of the TP agonists U46619 and IBOP or the isoprostanes iPE2III and iPF2αIII for 10 min. cAMP was quantified as described under "Experimental Procedures." Data are presented as mean pmol of cAMP/well ± S.E. from four experiments, each performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Effect of TP activation on cAMP generation in TPα-HEK and IP/TPα-HEK. U46619 (A) or iPE2III (B) was applied to TPα-HEK (closed squares), IP-HEK (closed triangles), or IP/TPα-HEK (closed circles) for 10 min, and cAMP was quantified as indicated under "Experimental Procedures." Data are presented as mean pmol cAMP per well ± S.E. from 3–5 experiments performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Effect of SQ 29548 on TP induced cAMP generation in ASMC. hASMC (A and B) or WT mASMC (open bar) and IPKO mASMC (closed bar) (C and D) were treated with IBOP (A and C) or iPE2III (B and D) for 10 min. E and F, hASMC were pretreated with (open bar) or without (closed bar) SQ 29548 (10 μm, 30 min), followed by IBOP (E) or iPE2III (F) for 10 min. cAMP was quantified as described under "Experimental Procedures." Data are presented as the mean pmol of cAMP/well ± S.E. from 3–5 experiments, each performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of IP Coactivation on TP-mediated cAMP Generation— Activation of IP in IP/TPα co-expressing cells, with a submaximal concentration of cicaprost (0.02 nm, 5 min), resulted in a synergistic enhancement of cAMP generation in response to the TP agonist U46619 and to iPE2III (Fig. 7). Furthermore, activation of IP synergistically enhanced iPE2III-induced cAMP generation in both hASMC and mASMC (Fig. 8, B and D). In contrast, whereas I
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