Introduction

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TP is a G protein-coupled receptor (Hirata et al., 1991) that, on ligand stimulation, results in activation of PLC and subsequent increases in inositol IP₃, DAG and Ca⁺⁺_i concentrations (Brass et al., 1987). The human TP cDNA was originally cloned from placenta and a platelet-like megakaryocyte cell line (Hirata et al., 1991). In a result consistent with previous pharmacologic and biochemical evidence pointing to the existence of TP isoforms (Dorn, 1989; Takahara et al., 1990; Furci et al., 1991), a second form of TP has recently been cloned from human umbilical vein endothelial cells (HUVECs) (Raychowdhury et al., 1994, 1995). The endothelial receptor, termed TP, and the platelet/placental TP, termed TP, are derived by an alternative splicing mechanism; they are identical for the first 328 amino acids but differ in their carboxyl terminal cytoplasmic tails. TP activity is regulated in vivo both by direct phosphorylation and by regulated expression of the TP gene (Kinsella et al., 1994a). Polymerase chain reaction (PCR) analyses have confirmed that HUVECs contain only TP, whereas both TP and TP are expressed in placental tissues and in platelets (Raychowdhury et al., 1994, 1995; Hirata et al., 1996; Miggin and Kinsella, unpublished). DNA sequence analysis has also pointed to sequence polymorphisms in the promoter of the gene coding for the human TPs (Kinsella et al., 1994b).

Using a variety of in vitro approaches, involving reconstitution studies (Shenker et al., 1991), copurification experiments (Knezevic et al., 1993) or photo cross-linking studies with GTP analogs (Offermanns et al., 1994), various investigators have previously proposed that the platelet TP(s) might couple to the heterotrimeric G proteins G_q and G₁₂. However, there has been no direct demonstration of functional coupling between the TP receptor and these G proteins in vivo.

The isoprostanes (O'Connor et al., 1984) are biologically potent prostanoids that are primarily generated in vivo by nonenzymatic, free radical-catalyzed lipid peroxidations (Morrow et al., 1990). D-ring, E-ring and F-ring isoprostanes are generated in vivo (Morrow et al., 1990; Morrow et al., 1994a, 1994b; Takahashi et al., 1992). One of these compounds, 8-epi PGF₂, may also be synthesized as a product of COX enzymes in human platelets and monocytes (Pratico and Fitzgerald, 1995; 1996). A potent vasoconstrictor in both lung and kidney, 8-epi PGF₂ significantly reduces renal blood flow and glomerular filtration rates (Morrow et al., 1990, Morrow et al., 1994a). In rabbits, the vasoconstriction of pulmonary vasculature induced by 8-epi PGF₂ appears to be due to the activation of the SQ29,548 responsive TP(s) (Banerjee et al., 1992). Whereas 8-epi PGF₂ induces a shape change in human platelets at 10⁶ M and 10⁵ M, at higher concentrations (10⁴ M) it induces reversible but not irreversible aggregation (Morrow et al., 1992). All of these actions of 8-epi PGF₂ were blocked by the TP antagonist SQ 29,548. However, both 8-epi PGF₂ and its structural isomers 9, 11-PGF₂ and PGF₂ also inhibit platelet aggregation induced by such PG endoperoxide/TXA₂ analogs as U46619, I-BOP {5-heptenoic acid, 7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo [2.2.1] hept-2-yl]-,[15-[1, 2(Z), 3(1E,3S*), 4]]} and by arachidonic acid. This suggests that 8-epi PGF₂ may function as an antagonist of the platelet TPs (Morrow et al., 1992; Yin et al., 1994). More recently, it has been shown that whereas 8-epi PGF₂ can inhibit platelet aggregation induced by low-dose collagen but not thrombin, it can also potentiate reversible platelet aggregation in response to low-dose ADP (Yin et al., 1994). This suggests that 8-epi PGF₂ may have partial agonist activity, mediated through the platelet TP(s) (Yin et al., 1994). Furthermore, it has been suggested that 8-epi PGF₂ may exert its biological actions in vascular smooth muscle through activation of receptor sites related to but distinct from TPs (Fukunaga et al., 1993). However, in competition binding studies, both we (Kinsella et al., 1994b; Pratico et al., 1996) and others (Yin et al., 1994) have shown that very high concentrations of 8-epi PGF₂ are necessary to displace the radiolabeled TXA₂ antagonist SQ 29,548 or the agonist I-BOP from either the cloned TP isoform expressed in human embryonic kidney 293 cells or the TPs expressed on platelets, 8-epi PGF₂ being approximately 1000 times less effective than the unlabeled analogs of either TXA₂ (I-BOP) or PGH₂ (U46619) or the antagonist SQ29,548 (Kinsella et al., 1994b). These observations, coupled with the discrepancies between the EC₅₀ values for ligand displacement and the concentrations of 8-epi PGF₂ in the circulation (Morrow et al., 1990) even in syndromes of oxidant stress, call into question the role of this compound as an endogenous TP ligand in vivo.

Thus, in order to establish directly whether the TP receptor isoform can couple to members of the heterotrimeric G_q family, resulting in activation of phospholipase C, we have coexpressed the human TP isoform and the  subunits of G_q or G₁₁ in HEK 293 cells, and have analyzed changes in Ca⁺⁺_i concentrations in FURA2/AM-loaded HEK 293 transfectants in response to stimulation by the stable TXA₂ mimetic U46619. HEK 293 cells express very low levels of endogenous TPs (Kinsella et al., 1994b) or G_q or G₁₁ (Conklin et al., 1992) and therefore provide an ideal background to define the individual components of the TP receptor-mediated signal transduction pathways. To address the question whether the isoprostane 8-epi PGF₂ can functionally activate the platelet/placental TP isoform, we have also analyzed the ability of this ligand to induce mobilization of Ca⁺⁺_i in transfected HEK 293 cells and in platelets. Given the recent study that demonstrated that receptor affinity for synthetic TP mimetics might be modulated by cotransfected G proteins G₁₃ and G_q (Allan et al., 1996), we also wished to address the possibility that G protein coexpression enhances the affinity of TP for 8-epi PGF₂, relative to the mimetic U46619. Our results demonstrate that the human TP isoform functionally couples to the G proteins G_q and G₁₁ in vivo and that 8-epi PGF₂ may indeed directly activate the cloned TP isoform expressed in the HEK 293 cells and also the TP receptor(s) expressed in platelets. Furthermore, although either TP receptor: G protein complex may be activated by 8-epi PGF₂, it remains a considerably less potent ligand than either U46619 or I-BOP, structural mimetics of PG endoperoxides and TXA₂.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Materials. The following chemicals were obtained from Cayman Chemical Company: 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo [2,2,1] hept-5-yl]-[1R-[1,4,5(z), 6(1E,3S*)]-9,11-dideoxy-9,11-methanoepoxy prostaglandin F₂ (U46619); 8-epi PGF₂; 5-heptenoic acid, 7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo [2.2.1] hept-2-yl]-,[15-[1, 2(Z), 3(1E,3S*), 4]] (I-BOP); 5-heptenoic acid, 7-[3-[[Z-[phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo [2.2.1] hept-2-yl]-,[1S-[1,2(Z),3,4]] (SQ29,548). Thrombin and [1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-2-oxy}-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid, pentaacetoxymethyl ester] (FURA2/AM) were obtained from Calbiochem. [³H]SQ29,548 (50.4 Ci/mmol) and anti G-protein GA/I antibody were obtained from DuPont NEN.

Plasmids. The plasmids p3:Gq and pBluescript(KS⁺):G11, coding for the  subunits of the mouse heterotrimeric G proteins G_q and G₁₁ (Strathman and Simon, 1990), respectively, were kindly donated by Dr. Melvin Simon, California Institute of Technology, Pasadena, CA. The plasmid pCMV:Gq was constructed by subcloning the full-length coding sequence for G_q from plasmid p3:Gq into the Kpn1-BamH 1 sites of pCMV5 (Andersson et al., 1989). The plasmid pCMV:G11 was constructed by subcloning the full-length coding sequence for G₁₁ from plasmid pBluescript(KS⁺):G11 into the Kpn1-HindIII sites of pCMV5. The plasmids pCMV5 and pCMVTXR, containing the full-length cDNA for the human platelet/placental TP, have been previously described (Andersson et al., 1989; Kinsella et al., 1994a, respectively).

Cell culture and transfections. HEK 293 cells, obtained from the American Type Culture Collection, were grown in minimal essential medium containing 10% heat-inactivated horse serum. For transfection studies, HEK 293 cells were plated in 100-mm culture dishes approximately 24 hr before transfection at a density of 1.8 to 2 × 10⁶ cells/dish. Cells were transfected with 10 µg of pADVA (Gorman et al., 1990) and 25 µg of pCMV5 or the pCMV-based vectors using the calcium phosphate/DNA coprecipitation procedure (Graham and van der Eb, 1973). The cells were harvested 48 hr after transfection.

Radioligand binding and Western blot analysis. Transfected HEK 293 cells were harvested by centrifugation at 500 × g at 4°C for 5 min, were washed twice in Dulbecco's phosphate-buffered saline (PBS) and were resuspended in modified Ca⁺⁺/Mg⁺⁺-free Hank's buffered salt solution containing 10 mM HEPES, pH 7.67, and 0.1% bovine serum albumin (HBSSHB buffer). Alternatively, in order to fractionate the cells into their soluble (S100) and membrane (P100) components, washed cells were resuspended and homogenized in HED buffer (20 mM HEPES, pH 7.67, 1 mM EGTA and 0.5 mM dithiothreitol) supplemented with 1 mM phenylmethylsulfonyl fluoride and 10 µM indomethacin. The homogenates were centrifuged at 100,000 × g for 30 min at 4°C, and the membrane fractions (P100) were resuspended in HEDG buffer (20 mM HEPES, pH 7.67, 1 mM EGTA, 0.5 mM dithiothreitol, 100 mM NaCl and 10% glycerol) supplemented with 1 mM phenylmethylsulfonyl fluoride and 10 µM indomethacin. Protein determinations were carried out according to the Bradford assay (Bradford, 1976). For ligand binding studies, protein concentrations in the membrane fractions and whole-cell fractions were diluted to 1 mg/ml in HEDG and HBSSHB buffer, respectively. Saturation radioligand binding experiments with the TP antagonist [³H] SQ 29,548 (20 nM, 50.4 Ci/mmol) were carried out at 30°C for 30 min in 100-µl reactions. Nonspecific binding was determined in the presence of excess nonlabeled SQ 29,548 (10 µM). Reactions were terminated by the addition of 4 ml of ice-cold 10 mM Tris-HCl, pH 7.4, followed by filtration through Whatman GF/C glass filters. Subsequent washing of the filters three times with 10 mM Tris-HCl, pH 7.4, was followed by liquid scintillation counting of the filters in 5 ml of scintillation fluid.

Preparation of platelets. Blood was drawn via venipuncture from normal human volunteers, who had not taken any medication for at least 10 days, into syringes containing indomethacin (10 µM) and 3.8% sodium citrate (9:1 v/v) (final concentration 0.38% sodium citrate). The blood was centrifuged for 10 min at 160 × g; the platelet-rich plasma (PRP) was removed and recentrifuged for 10 min at 160 × g to remove contaminating red blood cells. Where necessary, PPP was prepared by spinning the remaining blood at 900 × g for 15 min. The quality of the PRP was routinely checked by monitoring the aggregation properties of the platelets (data not shown). For aggregation studies, platelets in PRP were diluted to approximately 10⁸ platelets/ml in PPP; 0.5-ml aliquots were preincubated at 37°C for 2 min before addition of the aggregating agent (1 µM U46619 or 0.1 U/ml thrombin), and the extent of aggregation was monitored by light transmission in a Biodata Pap 4 aggregometer.

Calcium measurements. Ca⁺⁺_i measurements either in transfected HEK 293 cells or in platelets were made by monitoring the intensity of FURA2 fluorescence. For the transfected cells, 48 hr after transfection the HEK 293 cells were washed twice in PBS, resuspended in HBSSHB buffer at 10⁷ cells/ml and incubated in the dark with 5 µM FURA2/AM for 45 min at 37°C. Subsequently the cells were collected by centrifugation (900 × g, 5 min), washed once in an equal volume of HBSSHB and finally resuspended in HBSSHB buffer at 10⁷ cells/ml and kept at room temperature in the dark until use. For each measurement of Ca⁺⁺_i, aliquots of HEK 293 cells were diluted to 0.825 × 10⁶ cells/ml in HBSSHB buffer containing 1 mM CaCl₂. For platelet studies, PRP was incubated in the dark with 5 µM FURA2/AM (Calbiochem) at 37°C for 45 min; platelets were harvested by centrifugation (900 × g, 15 min), washed once in resuspension buffer (10 mM HEPES, 145 mM NaCl, 5 mM KCl, 5.5 mM glucose, pH 7.4) and finally resuspended in resuspension buffer containing 1 mM CaCl₂ at a cell density of 3 × 10⁸/ml and kept at room temperature in the dark until use.

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	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Coexpression of the TP isoform and G_q or G₁₁ in transfected HEK 293 cells. To address directly whether it might couple to the heterotrimeric G proteins G_q and/or G₁₁, we transiently expressed the TP isoform in HEK 293 cells, either in the presence of the  subunits of the heterotrimeric G protein G_q or G₁₁ or in the presence of the control vector pCMV5. Expression of TP was confirmed by radioligand binding assays using the radiolabeled TP antagonist [³H]SQ29,548 (Ogletree et al., 1985). G-protein expression was confirmed by Western blot analysis using an antibody, GA/I, that recognizes a conserved region within many heterotrimeric G-protein  subunits (Goldsmith et al., 1988). In line with our previous reports (Kinsella et al., 1994a), HEK 293 cells display very low levels of TP expression (66 ± 12.9 fmol/mg protein, table 1); thus they provide an ideal background in which to study activation of the transfected TP. Transfection of HEK 293 cells with the TP cDNA (pCMVTXR) either in the presence of the control vector, pCMV5, or in the presence of plasmids coding for either G_q (pCMV:Gq) or G₁₁ (pCMV:G11) resulted in high-level TP expression (table 1) as compared with the level of TP radioligand binding in cells transfected with the control vector only (table 1). Positive expression of the heterotrimeric G proteins G_q and G₁₁ was observed in the membrane fractions of cells transfected with the corresponding cDNAs coding for G_q and G₁₁, respectively, but not in the control transfected cells or in those cells transfected with TP only (fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   HEK 293 cells were transfected with the plasmids pCMV5 (lanes 1 and 2); pCMV5 and pCMVTXR (lanes 3 and 4); pCMV:Gq and pCMVTXR (lanes 5 and 6); pCMV:G11 and pCMVTXR (lanes 7 and 8). At 48 hr after transfection, the cells were harvested and fractionated into their soluble (S100; lanes 1, 3, 5 and 7) and particulate (P100, lanes 2, 4, 6 and 8) components and were then subjected to SDS-PAGE and Western blot analysis using the anti G-protein GA/I antibody (1:2000). That region of the blot corresponding to proteins of molecular weight between 30 and 50 kDa is shown.

U46619 and 8-epi PGF₂ induce Ca⁺⁺_i mobilization in HEK 293 cells cotransfected with the cDNAs for the TP isoform and G_q or G₁₁. Functional coupling of TP to PLC activation was assessed throughout by monitoring mobilization of Ca⁺⁺_i in FURA/2AM-loaded cells in response to TP selective ligand U46619 or the isoprostane 8-epi PGF₂. Stimulation of HEK 293 cells, transfected with either the vector pCMV5 control or TP, with 10⁶ M U46619 (fig. 2A and B, respectively) or with 10⁵ M 8-epi PGF₂ (fig. 3, A and B, respectively) failed to induce a transient rise in Ca⁺⁺_i. In contrast, stimulation of HEK 293 cells cotransfected with either TP and G_q or TP and G₁₁ by either 10⁶ M U46619 (fig. 2C; [Ca⁺⁺]_i = 54.15 ± 8.4 nM and fig. 2D; [Ca⁺⁺]_i = 107.6 ± 22.6 nM, respectively) or 10⁵ M 8-epi PGF₂ (fig. 3C; [Ca⁺⁺]_i = 53.2 ± 14.4 nM and fig. 3D; [Ca⁺⁺]_i = 99.4 ± 49.2 nM, respectively) resulted in a rapid, transient rise in Ca⁺⁺_i levels.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Transfected HEK 293 cells were preloaded with FURA2/AM and were stimulated with the TP receptor mimetic U46619 (1 µM) at the times indicated by the arrows. In the specific panels, HEK 293 cells were transfected with: A) pCMV5 control vector only; B) pCMV5 and pCMVTXR; C) pCMVTXR and pCMV:Gq; D) pCMVTXR and pCMV:G11. FURA2/AM fluorescence was recorded on a Perkin-Elmer LS50-B spectrofluorimeter at excitation wavelengths of 340 nM and 380 nM and emission wavelengths of 510 nM. The ratio of fluorescence at 340 nM to fluorescence at 380 nM is a measure of Ca++i mobilization, assuming a Kd of 225 nM Ca++ for FURA2/AM. The results presented are representative data from at least four independent experiments and are plotted as changes in intracellular Ca++ concentrations mobilized ([Ca++]i ± S.E. (nM)) as a function of time (seconds) on ligand stimulation. Levels of intracellular Ca++ mobilized were: C) [Ca++]i = 54.15 ± 8.4 nM (n = 4), D) [Ca++]i = 107.6 ± 22.6 nM (n = 4).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Transfected HEK 293 cells were preloaded with FURA2/AM and were then stimulated with 8-epi PGF2 (10 µM) at the times indicated by the arrows. In the specific panels, HEK 293 cells were transfected with: A) pCMV5; B) pCMV5 and pCMVTXR; C) pCMVTXR and pCMV:Gq; D) pCMVTXR and pCMV:G11. FURA2/AM fluorescence was recorded on a Perkin-Elmer LS50-B spectrofluorimeter at excitation wavelengths of 340 nM and 380 nM and emission wavelengths of 510 nM. The ratio of fluorescence at 340 nM to fluorescence at 380 nM is a measure of Ca++i mobilization, assuming a Kd of 225 nM Ca++ for FURA2/AM. The results presented are representative data from at least four independent experiments and are plotted as changes in intracellular Ca++ concentrations mobilized ([Ca++]i ± S.E. (nM)) as a function of time (seconds) upon ligand stimulation. Levels of intracellular Ca++ mobilized were: C) [Ca++]i = 53.18 ± 14.4 nM (n = 4); D) [Ca++]i = 99.4 ± 49.2 nM (n = 4).

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View larger version (20K): [in this window] [in a new window]   Fig. 4.   HEK 293 cells, transfected with the plasmids pCMVTXR and pCMV:G11, were preloaded with FURA2/AM and were stimulated with the following agents at the times indicated by the arrows: A) 1 µM U46619, 80 sec and 180 sec; B) 10 µM 8-epi PGF2 at 40 sec and 160 sec; C) 1 µM U46619 at 35 sec, followed by 10 µM 8-epi PGF2 at 180 sec; D) 10 µM 8-epi PGF2 at 35 sec, followed by 1 µM U46619 at 190 sec; E) 10 µm SQ29,548 at 30 sec, followed by 1 µm U46619 at 180 sec. FURA2/AM fluorescence was recorded on a Perkin-Elmer LS50-B spectrofluorimeter at excitation wavelengths of 340 nM and 380 nM and emission wavelengths of 510 nM. The ratio of fluorescence at 340 nM to fluorescence at 380 nM is a measure of Ca++i mobilization, assuming a Kd of 225 nM Ca++ for FURA2/AM. The results presented are representative data from at least four independent experiments and are plotted as changes in intracellular Ca++ concentrations mobilized ([Ca++]i ± S.E. (nM)) as a function of time (seconds) upon ligand stimulation. Levels of intracellular Ca++ mobilized were: A) [Ca++]i = 98.3 ± 32.8 nM (n = 4); B) [Ca++]i = 67.75 ± 15.5 nM (n = 4); C) [Ca++]i = 100.3 ± 28.6 nM (n = 4); D) [Ca++]i = 74 ± 20.2 nM (n = 4).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   HEK 293 cells, transfected with the plasmids pCMVTXR and pCMV:G11, were preloaded with FURA2/AM and were stimulated with the following agents at the times indicated by the arrows: A) thrombin (0.1 U/ml) at 15 sec, followed by 1 µm U46619 at 140 sec; B) 1 µm U46619 at 45 sec, followed by thrombin (0.1 U/ml) at 145 sec. FURA2/AM fluorescence was recorded on a Perkin-Elmer LS50-B spectrofluorimeter at excitation wavelengths of 340 nM and 380 nM and emission wavelengths of 510 nM. The ratio of fluorescence at 340 nM to fluorescence at 380 nM is a measure of Ca++i mobilization, assuming a Kd of 225 nM Ca++ for FURA2/AM. The results presented are representative data from at least four independent experiments and are plotted as changes in intracellular Ca++ concentrations mobilized ([Ca++]i ± S.E. (nM)) as a function of time (seconds) upon ligand stimulation. Levels of intracellular Ca++ mobilized were: A) [Ca++]i = 120 ± 16.4 nM (n = 4) for thrombin, [Ca++]i = 113.1 ± 41.7 nM (n = 4) for U46619; B) [Ca++]i = 139.1 ± 74.4 nM (n = 4) for U46619, [Ca++]i = 130 ± 19.5 nM (n = 4) for thrombin.

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8-epi PGF₂ induces Ca⁺⁺_i mobilization in platelets through activation of TP. Stimulation of FURA2/AM-loaded platelets with the TP agonist U46619 (10⁶ M) induced a rapid, transient rise in Ca⁺⁺_i (fig. 6A; [Ca⁺⁺]_i = 210.6 ± 25.9 nM). Stimulation of platelets with 8-epi PGF₂ (10⁵ M) also induced a rapid, transient rise in Ca⁺⁺_i levels (fig. 6B; [Ca⁺⁺]_i = 58.8 ± 13.5 nM). Preincubation of platelets with SQ29,548 (10⁶ M) blocked Ca⁺⁺_i mobilization in response to subsequent stimulation with either 10⁶ M U46619 (fig. 6C) or 10⁵ M 8-epi PGF₂ (fig. 6D). On the other hand, initial stimulation of platelets with 10⁶ M U46619, which resulted in the transient rise in [Ca⁺⁺]_i, desensitized a subsequent rise in Ca⁺⁺_i upon treatment with 10⁶ M U46619 (fig. 7A; [Ca⁺⁺]_i = 222.5 ± 28.8 nM). Similarly, initial activation of platelets with 10⁵ M 8-epi PGF₂, which resulted in the elevation of Ca⁺⁺_i levels, also desensitized a subsequent rise in Ca⁺⁺_i upon treatment with 8-epi PGF₂ (fig. 7B; [Ca⁺⁺]_i = 61.25 ± 17.2 nM). By the same token, pretreatment with 10⁶ M U46619 (fig. 7C; [Ca⁺⁺]_i = 210 ± 35.14 nM) or 10⁵ M 8-epi PGF₂ (fig. 7D; [Ca⁺⁺]_i = 61.75 ± 14.8 nM) desensitized a secondary rise in Ca⁺⁺_i upon stimulation with 10⁵ M 8-epi PGF₂ or 10⁶ M U46619 (fig. 7, C and D, respectively), whereas initial stimulation of platelets with thrombin (0.1 U/ml) did not desensitize secondary stimulation with either U46619 (fig. 7E; [Ca⁺⁺]_i = 402 ± 49.6 nM for thrombin, [Ca⁺⁺]_i = 223.2 ± 26.35 nM for U46619) or 8-epi PGF₂ (data not shown). Thus, in agreement with previously published data, pretreatment of platelets with the TP antagonist SQ 29,548 abolished the rise in Ca⁺⁺_i induced by either U46619 or 8-epi PGF₂ (data not shown). This indicates that the rise in Ca⁺⁺_i induced by 8-epi PGF₂ at these concentrations is mediated by activation of platelet TP(s). Moreover, U46619, a known agonist of platelet TP(s), cross-desensitized the rise in Ca⁺⁺_i induced by 8-epi PGF₂. Similarly, primary stimulation with 8-epi PGF₂ cross-desensitized the U46619-induced elevation of Ca⁺⁺_i, which is substantial evidence of the activation of platelet TP by the isoprostane 8-epi PGF₂. Similar results were obtained for the TP agonist I-BOP, a closer structural mimetic of TXA₂ than U46619 (Dorn, 1989). I-BOP induced a rapid, transient rise in Ca⁺⁺_i that rapidly desensitized a secondary rise in Ca⁺⁺_i in response to I-BOP, U46619 or 8-epi PGF₂; 8-epi PGF₂ almost completely desensitized a second rise in Ca⁺⁺_i in response to I-BOP (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Platelets were preloaded with FURA2/AM and were stimulated with the following agents at the times indicated by the arrows: A) 1 µM U46619, 30 sec; B) 10 µM 8-epi PGF2, 20 sec; C) 1 µM SQ29,548, 20 sec, followed by 1 µM U46619, 180 sec; D) 1 µM SQ29,548, 20 sec, followed by 10 µM 8-epi PGF2, 160 sec. FURA2/AM fluorescence was recorded on a Perkin-Elmer LS50-B spectrofluorimeter at excitation wavelengths of 340 nM and 380 nM and emission wavelengths of 510 nM. The ratio of fluorescence at 340 nM to fluorescence at 380 nM is a measure of Ca++i mobilization, assuming a Kd of 225 nM Ca++ for FURA2/AM. The results presented are representative data from at least four independent experiments and are plotted as changes in intracellular Ca++ concentrations mobilized ([Ca++]i ± S.E. (nM)) as a function of time (seconds) upon ligand stimulation. Levels of intracellular Ca++ mobilized were: A) [Ca++]i = 210.6 ± 25.9 nM (n = 4); B) [Ca++]i = 58.8 ± 13.5 nM (n = 4).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Platelets were preloaded with FURA2/AM and were stimulated with the following agents at the times indicated by the arrows: A) 1 µM U46619, 30 sec and 140 sec; B) 10 µM 8-epi PGF2, 20 sec and 130 sec; C) 1 µM U46619, 35 sec, followed by 10 µM 8-epi PGF2 at 170 sec; D) 10 µM 8-epi PGF2 at 25 sec, followed by 1 µM U46619 at 165 sec; E) thrombin (0.1 U/ml) at 40 sec, followed by 1 µm U46619 at 160 sec. FURA2/AM fluorescence was recorded on a Perkin-Elmer LS50-B spectrofluorimeter at excitation wavelengths of 340 nM and 380 nM and emission wavelengths of 510 nM. The ratio of fluorescence at 340 nM to fluorescence at 380 nM is a measure of Ca++i mobilization, assuming a Kd of 225 nM Ca++ for FURA2/AM. The results presented are representative data from at least four independent experiments and are plotted as changes in intracellular Ca++ concentrations mobilized ([Ca++]i ± S.E. (nM)) as a function of time (seconds) upon ligand stimulation. Levels of intracellular Ca++ mobilized were: A) [Ca++]i = 222.5 ± 28.8 nM (n = 4); B) [Ca++]i = 61.25 ± 17.2 nM (n = 4); C) [Ca++]i = 210 ± 35.14 nM (n = 4); D) [Ca++]i = 61.75 ± 14.8 nM (n = 4); E) [Ca++]i = 402 ± 49.6 nM (n = 4) for thrombin, [Ca++]i = 223.2 ± 26.35 nM (n = 4) for U46619.

U46619 induces mobilization of Ca⁺⁺ from intracellular stores. To demonstrate that the Ca⁺⁺ mobilized in platelets and the transfected HEK 293 cells in response to TP activation was of intracellular origin, before agonist stimulation we preincubated FURA2/AM-loaded cells with EGTA in order to chelate extracellular Ca⁺⁺. Preincubation of platelets with EGTA did not interfere with the elevation of Ca⁺⁺_i levels upon stimulation with U46619 (compare figs. 8A and 8B, [Ca⁺⁺]_i = 208.9 ± 25 nM vs. fig. 8B, [Ca⁺⁺]_i = 209.75 ± 41.5 nM). Similar results were observed in HEK 293 cells transfected with TP and G_q (fig. 8C, [Ca⁺⁺]_i = 128 ± 35.3 nM). Taken together, these results demonstrate that U46619 induces the mobilization of Ca⁺⁺_i stores after activation of TP.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Platelets (panels A and B) or transfected HEK 293 cells (pCMVTXR and pCMV:Gq, panel C) were preloaded with FURA2/AM and were treated with the following agents at the indicated time-points: A) 1 µM U46619 at 18 sec; B) 1 mM EGTA at 30 sec, followed by 1 µM U46619 at 90 sec; C) 1 mM EGTA at 20 sec, followed by 1 µM U46619 at 65 sec. FURA2/AM fluorescence was recorded on a Perkin-Elmer LS50-B spectrofluorimeter at excitation wavelengths of 340 nM and 380 nM and emission wavelengths of 510 nM. The ratio of fluorescence at 340 nM to fluorescence at 380 nM is a measure of Ca++i mobilization, assuming a Kd of 225 nM Ca++ for FURA2/AM. The results presented are representative data from at least four independent experiments and are plotted as changes in intracellular Ca++ concentrations mobilized ([Ca++]i ± S.E. (nM)) as a function of time (seconds) upon ligand stimulation. Levels of intracellular Ca++ mobilized were: A) [Ca++]i = 208.9 ± 25 nM (n = 4); B) [Ca++]i = 209.75 ± 41.5 nM (n = 4); C) [Ca++]i = 128 ± 35.3 nM (n = 4).

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

We have investigated the functional coupling of the human placental/platelet TP isoform to the heterotrimeric G proteins G_q and G₁₁ in transfected HEK 293 cells by monitoring activation of the downstream signal transduction events as reflected by elevation of Ca⁺⁺_i levels after activation with the prostaglandin endoperoxide/TXA₂ mimetic U46619. In addition, we have explored the structure-function relationships of receptor-G protein action by comparing the effects of U46619 and the isoprostane 8-epi PGF₂ on the platelet TP(s) and on the cloned platelet/placental TP (Hirata et al., 1991), expressed in the mammalian HEK 293 cells. U46619 induced a rapid, transient rise in Ca⁺⁺_i in platelets and in HEK 293 cells cotransfected with TP and G_q or G₁₁. It is noteworthy that in the transfected HEK 293 cells, we did not observe a significant rise in Ca⁺⁺_i in response to U46619 unless the TP was coexpressed in the presence of a member of the G_q subfamily of heterotrimeric G proteins. It has been reported that HEK 293 cells lack endogenous G_q (Conklin et al., 1992) and that alpha-2 adrenoreceptor stimulation of phosphoinositide-specific PLC (PI-PLC) activity in HEK 293 cells was completely dependent on the coexpression of G_q (Conklin et al., 1992). Using a variety of in vitro approaches involving reconstitution studies (Shenker et al., 1991), copurification experiments (Knezevic et al., 1993) or cross-linking studies with photoactivated GTP analogs (Offermanns et al., 1994) various investigators have previously proposed that TP might couple to G_q and G₁₂. Our studies in the transfected HEK 293 cells now directly demonstrate that the platelet/placental TP isoform can functionally couple to G_q or to G₁₁ in vivo, resulting in the downstream mobilization of Ca⁺⁺_i after stimulation with U46619. In HEK 293 cells, cotransfection of G₁₁ produced greater mobilization of Ca⁺⁺_i than cotransfection of G_q in response to U46619 stimulation, which suggests a possible preference by TP for G₁₁ or the preferential coupling of G₁₁ vs. G_q to PLC in this system. In this study, we have also directly established that the isoprostane 8-epi PGF₂ induced Ca⁺⁺_i mobilization in HEK 293 cells cotransfected with TP and G_q or G₁₁; cotransfection of G₁₁ produced greater mobilization of Ca⁺⁺_i than cotransfection of G_q in response to 8-epi PGF₂ stimulation. This again indicates a possible preference by TP for G₁₁ in this system. 8-epi PGF₂ failed to mobilize Ca⁺⁺ in HEK 293 cells transfected with the TP receptor alone or with the vector control only. In HEK cotransfected cells and in platelets, higher concentrations of 8-epi PGF₂ (10⁵ M) than of U46619 (10⁶) were required to evoke a half-maximal response in either cell type; however, the maximal Ca⁺⁺ response observed in platelets was 3- to 4-fold greater after stimulation with U46619 than with 8-epi PGF₂.

The initial response to U46619 or to 8-epi PGF₂ rapidly desensitized a second rise in Ca⁺⁺_i levels in response to subsequent stimulation by either agent in platelets and in transfected HEK 293 cells. Preincubation of platelets or transfected cells with thrombin, on the other hand, did not desensitize the rise in intracellular Ca⁺⁺ on subsequent stimulation with either U46619 or 8-epi PGF₂. Thus TP(s) in platelets or the TP isoform expressed in HEK 293 cells may be subject to homologous desensitization after activation by either U46619 or 8-epi PGF₂. The addition of 8-epi PGF₂ (10⁵ M) and U46619 (10⁶ M) together did not potentiate or antagonize the maximal level of Ca⁺⁺ mobilized in either platelets or transfected HEK 293 cells, which suggests that in platelets at least, 8-epi PGF₂ and U46619 may activate the same TP receptors. Moreover, the TP antagonist SQ29,548 was equipotent in abolishing the Ca⁺⁺ response in both platelets and transfected HEK 293 cells on stimulation with either U46619 or 8-epi PGF₂.

Although 8-epi PGF₂ may activate TPs, it is unknown whether this effect is of biological significance. Indeed, we have recently reported that the EC₅₀ concentrations for functional responses in platelets are much higher than the plasma concentrations of the ligand obtained, even in syndromes of oxidant stress (Pratico et al., 1996). The comparative potency of U46619 and 8-epi PGF₂ in platelets suggests that in this endogeneous system, 8-epi PGF₂ is likely to activate TPs in vivo only when produced in very large amounts as an incidental ligand, or perhaps it has a stronger affinity for a closely related but distinct receptor that has yet to be identified. In conventional terms, whereas the dose-response data do not favor 8-epi PGF₂ as an endogeneous TP ligand, receptor activation by 8-epi PGF₂ might occur if the ligand were presented via an unusual concentrated delivery system, such as microvesicles shed from activated cells, or through selective reincorporation of released isoprostanes into the membrane (Barry et al., 1996).

The transfected HEK system also addresses the capacity of the TP receptor to couple with different G proteins in a defined system. Whereas TP readily couples to both G_q and G₁₁ in the transfected cells, we cannot directly extrapolate the actual preference exhibited in an endogeneous system, such as platelets, where both TP isoforms and several G proteins are available. Recently, Allan et al. (1996) have reported that the affinity of the TP isoform for structural analogs of PG endoperoxides and TXA₂ might be modified, depending on the nature of a cotransfected G protein. However, cotransfection of G_q or G₁₁ did not appear markedly to increase the affinity of TP for 8-epi PGF₂, which remained a less-favored ligand than U46619 in all of our experimental conditions.

In summary, we have demonstrated that human TP functionally couples to members of the G_q family of heterotrimeric G proteins in vivo. Activation of the receptor-G-protein complexes was demonstrated using two structurally distinct ligands, U46619 and 8-epi PGF₂. Although generation of the latter by the COX 1 and COX 2 enzymes lends credence to the possibility that it may function as an autocoid, its biological role in vivo remains to be elucidated. Irrespective of this possibility, we have demonstrated that TPs in human platelets and the TP isoform may be specifically activated by this compound. Thus, although 8-epi PGF₂ represents a potential alternative endogenous ligand for this receptor, it is a considerably less avid ligand than either U46619 or I-BOP, structural mimetics of PG endoperoxides and TXA₂. Furthermore, it remains to be established whether incidental activation of TP receptors by 8-epi PGF₂ may actually contribute to the adverse effects of oxidant stress in vivo.