The Lysophosphatidic Acid Type 2 Receptor Is Required for Protection Against Radiation-Induced Intestinal Injury
2007; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.03.038
ISSN1528-0012
AutoresWenlin Deng, E Shuyu, Ryoko Tsukahara, William J. Valentine, Gangadhar G. Durgam, Veeresa Gududuru, Louisa Balázs, Venkatraman Manickam, Marcello Arsura, Lester VanMiddlesworth, Leonard R. Johnson, Abby L. Parrill, Duane D. Miller, Gábor Tigyi,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoBackground & Aims: We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo. Methods: The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA1 and LPA2 knockout mice exposed to different apoptotic stimuli. Results: OTP was more efficacious than LPA in reducing gamma irradiation–, camptothecin-, or tumor necrosis factor α/cycloheximide–induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3–positive cells but was ineffective in LPA2 knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA2 knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)100/30 radiation by 50%. Conclusions: Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype. Background & Aims: We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo. Methods: The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA1 and LPA2 knockout mice exposed to different apoptotic stimuli. Results: OTP was more efficacious than LPA in reducing gamma irradiation–, camptothecin-, or tumor necrosis factor α/cycloheximide–induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3–positive cells but was ineffective in LPA2 knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA2 knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)100/30 radiation by 50%. Conclusions: Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype. The stem cells of the intestinal mucosa represent one of the most radiation-vulnerable cell types in the mammalian body.1Potten C.S. Radiation, the ideal cytotoxic agent for studying the cell biology of tissues such as the small intestine.Radiat Res. 2004; 161: 123-136Crossref PubMed Scopus (203) Google Scholar Whereas free radical scavengers can ameliorate the central nervous system syndrome and the widely used bone marrow transplantation can effectively treat the hematopoietic syndrome caused by exposure to lethal doses of radiation, effectively treating the gastrointestinal syndrome due to radiation-induced apoptosis of the intestinal stem cells is more difficult. Lysophosphatidic acid (1-radyl-2-hydroxy-sn-glycero-3-phosphate; LPA) is a growth factor–like lipid mediator with antiapoptotic actions elicited through a set of G protein–coupled receptors.2Tigyi G. Parrill A.L. Molecular mechanisms of lysophosphatidic acid action.Prog Lipid Res. 2003; 42: 498-526Crossref PubMed Scopus (157) Google Scholar, 3Moolenaar W.H. van Meeteren L.A. Giepmans B.N. The ins and outs of lysophosphatidic acid signaling.Bioessays. 2004; 26: 870-881Crossref PubMed Scopus (499) Google Scholar At least 5 LPA G protein–coupled receptors have been identified so far.4Ishii I. Fukushima N. Ye X. Chun J. Lysophospholipid receptors: signaling and biology.Annu Rev Biochem. 2004; 73: 321-354Crossref PubMed Scopus (681) Google Scholar The LPA1, LPA2, and LPA3 receptors are encoded by the endothelial differentiation gene (EDG) family and share approximately 60% identity with each other.2Tigyi G. Parrill A.L. Molecular mechanisms of lysophosphatidic acid action.Prog Lipid Res. 2003; 42: 498-526Crossref PubMed Scopus (157) Google Scholar, 4Ishii I. Fukushima N. Ye X. Chun J. Lysophospholipid receptors: signaling and biology.Annu Rev Biochem. 2004; 73: 321-354Crossref PubMed Scopus (681) Google Scholar LPA4 and LPA5 are distantly related to the EDG family, share only 20%–24% amino acid identity with the EDG family, and functionally are less well characterized.5Noguchi K. Ishii S. Shimizu T. Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family.J Biol Chem. 2003; 278: 25600-25606Crossref PubMed Scopus (488) Google Scholar, 6Kotarsky K. Boketoft A. Bristulf J. Nilsson N.E. Norberg A. Hansson S. Owman C. Sillard R. Leeb-Lundberg L.M. Olde B. 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LPA2 receptor mediates mitogenic signals in human colon cancer cells.Am J Physiol Cell Physiol. 2005; 289: C2-C11Crossref PubMed Scopus (103) Google Scholar Each individual LPA receptor has its distinct coupling pattern to G proteins. LPA1 couples to Gi, Gq, and G12/13; LPA2 couples to Gi, Gq, and G12/13; and LPA3 couples to only Gi and Gq, not G12/13.10Ishii I. Contos J.J.A. Fukushima N. Chun J. Functional comparisons of the lysophosphatidic acid receptors, LPA1/VZG-1/EDG-2, LPA2/EDG-4, and LPA3/EDG-7 in neuronal cell lines using a retrovirus expression system.Mol Pharmacol. 2000; 58: 895-902Crossref PubMed Scopus (185) Google Scholar These G proteins share many downstream signaling pathways that act in a cooperative manner.11Yart A. Chap H. Raynal P. Phosphoinositide 3-kinases in lysophosphatidic acid signaling: regulation and cross-talk with the Ras/mitogen-activated protein kinase pathway.Biochim Biophys Acta. 2002; 1582: 107-111Crossref PubMed Scopus (59) Google Scholar The activation of the phosphoinositide 3-kinase (PI3K)-AKT and MEK-ERK1/2 prosurvival pathways has been well delineated in mediating LPA-initiated antiapoptotic activity.12Deng W. Balazs L. Wang D.A. Van Middlesworth L. Tigyi G. Johnson L.R. Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation- and chemotherapy-induced apoptosis.Gastroenterology. 2002; 123: 206-216Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 13Deng W. Wang D.A. Gosmanova E. Johnson L.R. Tigyi G. LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G821-G829PubMed Google Scholar, 14Ye X. Ishii I. Kingsbury M.A. Chun J. Lysophosphatidic acid as a novel cell survival/apoptotic factor.Biochim Biophys Acta. 2002; 1585: 108-113Crossref PubMed Scopus (117) Google Scholar LPA activates AKT, which in turn phosphorylates BAD and procaspase 9, leading to inhibition of apoptosis.15Kang Y.C. Kim K.M. Lee K.S. Namkoong S. Lee S.J. Han J.A. Jeoung D. Ha K.S. Kwon Y.G. Kim Y.M. Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation.Cell Death Differ. 2004; 11: 1287-1298Crossref PubMed Scopus (70) Google Scholar LPA-induced activation of mitogen-activated protein kinase/extracellular signal–regulated kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) 1/2 signaling can also activate BAD phosphorylation and attenuate caspase-9.16Fang X. Yu S. Eder A. Mao M. Bast Jr, R.C. Boyd D. Mills G.B. Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway.Oncogene. 1999; 18: 6635-6640Crossref PubMed Scopus (239) Google Scholar LPA has been shown to activate nuclear factor κB, which regulates important prosurvival genes.16Fang X. Yu S. Eder A. Mao M. Bast Jr, R.C. Boyd D. Mills G.B. Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway.Oncogene. 1999; 18: 6635-6640Crossref PubMed Scopus (239) Google Scholar, 17Chou C.H. Wei L.H. Kuo M.L. Huang Y.J. Lai K.P. Chen C.A. Hsieh C.Y. Up-regulation of interleukin-6 in human ovarian cancer cell via a Gi/PI3K-Akt/NF-kappaB pathway by lysophosphatidic acid, an ovarian cancer-activating factor.Carcinogenesis. 2005; 26: 45-52Crossref PubMed Scopus (78) Google Scholar A specific cellular response, including the antiapoptotic effect of LPA, might be mediated through a single LPA receptor or a combination of multiple receptor subtypes, which appear to be coexpressed in most cell types.18Fischer D.J. Liliom K. Guo Z. Nusser N. Virag T. Murakami-Murofushi K. Kobayashi S. Erickson J.R. Sun G. Miller D.D. Tigyi G. 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Virag T. Wada A. Yatomi Y. Kobayashi T. Igarashi Y. Tigyi G. Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood.J Biol Chem. 2002; 277: 21197-21206Crossref PubMed Scopus (226) Google Scholar High levels of phospholipids have been detected in the colonic mucosa of patients with inflammatory bowel disease, and LPA significantly reduces the degree of inflammation and necrosis in a rat model of colitis.27Sano T. Baker D. Virag T. Wada A. Yatomi Y. Kobayashi T. Igarashi Y. Tigyi G. Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood.J Biol Chem. 2002; 277: 21197-21206Crossref PubMed Scopus (226) Google Scholar LPA has been shown to stimulate restitution of intestinal epithelia via pertussis toxin (PTX)-sensitive mechanisms.28Hines O.J. Ryder N. Chu J. McFadden D. Lysophosphatidic acid stimulates intestinal restitution via cytoskeletal activation and remodeling.J Surg Res. 2000; 92: 23-28Abstract Full Text PDF PubMed Scopus (41) Google Scholar LPA2 receptors play an important attenuating role in bacterial toxin–induced secretory diarrhea via PDZ domain–mediated protein-protein interactions, inhibiting the activation of the CFTR Cl− channel.8Li C. Dandridge K.S. Di A. Marrs K.L. Harris E.L. Roy K. Jackson J.S. Makarova N.V. Fujiwara Y. Farrar P.L. Nelson D.J. Tigyi G.J. Naren A.P. Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.J Exp Med. 2005; 202: 975-986Crossref PubMed Scopus (130) Google Scholar Therefore, the food-derived LPA in the lumen and its receptors on the epithelium suggest a physiologic role for LPA in maintaining gastrointestinal integrity that can be explored for therapeutic intervention. Apoptosis in the intestinal epithelium is the primary pathologic factor that leads to chemotherapy- or radiation-induced gastrointestinal damage.1Potten C.S. Radiation, the ideal cytotoxic agent for studying the cell biology of tissues such as the small intestine.Radiat Res. 2004; 161: 123-136Crossref PubMed Scopus (203) Google Scholar, 29Hall P.A. Coates P.J. Ansari B. Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis.J Cell Sci. 1994; 107: 3569-3577Crossref PubMed Google Scholar, 30Pritchard D.M. Watson A.J. Apoptosis and gastrointestinal pharmacology.Pharmacol Ther. 1996; 72: 149-169Crossref PubMed Scopus (66) Google Scholar We formulated a hypothesis that LPA can be used for the protection of the mucosa from iatrogenic traumas (radiation injury in particular); if so, it could serve as a template for prosurvival drugs against radiation injury. This hypothesis is based on several lines of our work reported over the past few years. We showed that LPA protected intestinal epithelia against irradiation-induced apoptosis both in vitro and in vivo,12Deng W. Balazs L. Wang D.A. Van Middlesworth L. Tigyi G. Johnson L.R. Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation- and chemotherapy-induced apoptosis.Gastroenterology. 2002; 123: 206-216Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar also showed in vitro that LPA achieved its antiapoptotic effects through the PTX-sensitive activation of the MEK-ERK1/2 and PI3K-AKT prosurvival pathways,13Deng W. Wang D.A. Gosmanova E. Johnson L.R. Tigyi G. LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G821-G829PubMed Google Scholar and established that the antiapoptotic effect, unlike the mitogenic and motogenic effects of LPA, did not require the transregulation of several tyrosine kinase receptors.31Deng W. Poppleton H. Yasuda S. Makarova N. Shinozuka Y. Wang D.A. Johnson L.R. Patel T.B. Tigyi G. Optimal lysophosphatidic acid-induced DNA synthesis and cell migration but not survival require intact autophosphorylation sites of the epidermal growth factor receptor.J Biol Chem. 2004; 279: 47871-47880Crossref PubMed Scopus (20) Google Scholar However, LPA is not an optimal drug candidate. Exogenous LPA is rapidly metabolized in the gastrointestinal tract. Because complex lipids, including phospholipids, are broken down to nonpolar intermediates that traverse the plasma membrane, the action of LPA is terminated by phospholipase- and lipase-mediated deacylation or (lipid) phosphatase-mediated dephosphorylation. While this mechanism rapidly renders LPA inactive, it also limits the effect of LPA on the receptors present on the luminal surface of the epithelium. LPA consists of a glycerol backbone with a hydroxyl group, a phosphate group, and a fatty acid or fatty alcohol chain. We have developed and experimentally validated computational models of the EDG family of LPA receptors32Parrill A.L. Wang D. Bautista D.L. Van Brocklyn J.R. Lorincz Z. Fischer D.J. Baker D.L. Liliom K. Spiegel S. Tigyi G. Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate.J Biol Chem. 2000; 275: 39379-39384Crossref PubMed Scopus (142) Google Scholar, 33Wang D. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. 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Liliom K. van Meeteren L.A. Moolenaar W.H. Wilke N. Siess W. Tigyi G. Miller D.D. Synthesis, structure-activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of autotaxin.J Med Chem. 2005; 48: 4919-4930Crossref PubMed Scopus (101) Google Scholar Therefore, we hypothesized that long-chain fatty alcohol thiophosphates mimic LPA at the EDG family receptors and at the same time are metabolically stabilized against phospholipase cleavage, which requires a glycerol backbone. Furthermore, thiophosphates tend to be poor substrates of lipid phosphatases and render them resistant to breakdown. These observations led us to explore the pharmacologic and biologic properties of octadecenyl thiophosphate (OTP) as an orally bioavailable, metabolically stabilized, nonabsorbing LPA mimic with radioprotective action in the gut. The present study set multiple objectives. First, we explored the pharmacologic properties of OTP by using computational and pharmacologic approaches and determined its resistance to lipase and lipid phosphatase cleavage. Next, we compared the antiapoptotic effect of OTP with that of LPA in IEC-6 cells in vitro. Third, using a receptor add-back, we examined which LPA receptor subtypes mediate survival signals to prevent radiation-induced apoptosis. Fourth, we examined whether orally applied OTP reduces radiation-induced apoptosis and caspase-3 activation and increases crypt survival in clonogenic assays conducted in C57BL/6 mice exposed to 15 Gy gamma irradiation. We also evaluated the OTP- and LPA-induced activation of those prosurvival signaling pathways in vivo that we had previously established in vitro. Finally, we tested whether intraperitoneal administration of OTP prevents death caused by LD100/30 gamma irradiation. We found that OTP mitigated radiation-induced death, protected intestinal epithelial cells from apoptosis in vitro and in vivo, and was significantly more effective compared with LPA. Both LPA and OTP reduced apoptosis and caspase-3 activation and increased crypt survival in wild-type and LPA1 KO mice; however, both were ineffective in LPA2 KO mice. Together, these data suggest that OTP is a highly effective intestinal radioprotective agent that targets LPA2 as a prosurvival receptor. LPA (oleoyl) was purchased from Avanti Polar Lipids (Alabaster, AL). LPA and OTP (synthesized as described by Durgam et al40Durgam G.G. Virag T. Walker M.D. Tsukahara R. Yasuda S. Liliom K. van Meeteren L.A. Moolenaar W.H. Wilke N. Siess W. Tigyi G. Miller D.D. Synthesis, structure-activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of autotaxin.J Med Chem. 2005; 48: 4919-4930Crossref PubMed Scopus (101) Google Scholar) were applied to cells complexed with fatty acid–free bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO) as previously described.39Virag T. Elrod D.B. Liliom K. Sardar V.M. Parrill A.L. Yokoyama K. Durgam G. Deng W. Miller D.D. Tigyi G. Fatty alcohol phosphates are subtype-selective agonists and antagonists of LPA receptors.Mol Pharmacol. 2003; 63: 1032-1042Crossref PubMed Scopus (71) Google Scholar Camptothecin and cycloheximide (CHX) were purchased from Sigma Chemical Co. Recombinant rat tumor necrosis factor (TNF)-α was purchased from BD PharMingen (San Diego, CA). PD98059 was purchased from Calbiochem (San Diego, CA). PTX and Ac-DEVD-pNA and Ac-LEHD-pNA colorimetric and Ac-IETD-AFC fluorescent caspase substrates were from Biomol Laboratories, Inc (Plymouth Meeting, PA). The following antibodies and sources were used: rabbit anti–caspase-3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), rabbit anti-active caspase-3 (Abcam, Inc, Cambridge, MA), mouse monoclonal anti-JNK1 (BD PharMingen), mouse monoclonal anti-phospho (Thr183/Tyr185)-JNK, rabbit anti-ERK1/2, rabbit anti-phospho-(Tyr202/Tyr204) ERK1/2, rabbit anti-AKT, rabbit anti-phospho-(Ser473)-AKT, rabbit anti–Bcl-2, monoclonal mouse anti–Bcl-XL (Cell Signaling, Inc, Beverly, MA), monoclonal mouse anti–phospho-(Thr180/Tyr182)-P38 (Promega, Madison, WI), and mouse monoclonal anti-actin (Calbiochem). Horseradish peroxidase–conjugated anti-rabbit and anti-mouse secondary antibody used for Western blotting was purchased from Sigma Chemical Co. Fluorescein isothiocyanate–labeled goat anti-rabbit immunoglobulin G was purchased from Molecular Probes (Eugene, OR). Normal goat serum and Vectashield Mounting Medium with DAPI were purchased from Vector Laboratories, Inc (Burlingame, CA). The detailed methods used to develop computational models of LPA1, LPA2, and LPA3 have been reported previously.33Wang D. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. A single amino acid determines ligand specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors.J Biol Chem. 2001; 276: 49213-49220Crossref PubMed Scopus (97) Google Scholar, 34Sardar V.M. Bautista D.L. Fischer D.J. Yokoyama K. Nusser N. Virag T. Wang D.A. Baker D.L. Tigyi G. Parrill A.L. Molecular basis for lysophosphatidic acid receptor antagonist selectivity.Biochim Biophys Acta. 2002; 1582: 309-317Crossref PubMed Scopus (78) Google Scholar, 36Fujiwara Y. Sardar V. Tokumura A. Baker D.L. Murakami-Murofushi K. Parrill A.L. Tigyi G. Identification of residues responsible for ligand recognition and regioisomeric selectivity of LPA receptors expressed in mammalian cells.J Biol Chem. 2005; 280: 35038-35050Crossref PubMed Scopus (78) Google Scholar Briefly, our validated model of the S1P1 receptor32Parrill A.L. Wang D. Bautista D.L. Van Brocklyn J.R. Lorincz Z. Fischer D.J. Baker D.L. Liliom K. Spiegel S. Tigyi G. Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate.J Biol Chem. 2000; 275: 39379-39384Crossref PubMed Scopus (142) Google Scholar, 33Wang D. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. A single amino acid determines ligand specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors.J Biol Chem. 2001; 276: 49213-49220Crossref PubMed Scopus (97) Google Scholar, 37Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Sphingosine 1-phosphate analogue recognition and selectivity at S1P4 within the endothelial differentiation gene family of receptors.Biochem J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar was used as a template for generating LPA receptor models. Homology model development was performed using the automated algorithm implemented in the MOE software program.41MOE. Chemical Computing Group, Montreal2002Google Scholar The best model was geometry optimized using the MMFF9442Halgren T.A. Merck molecular force field I. Basis, form, scope, parameterization, and performance of MMFF94*.J Comp Chem. 1996; 17: 490-519Crossref Scopus (4584) Google Scholar force field to a root mean square gradient of 0.1 kcal/mol · Å. The individual receptor models were used in docking studies with OTP bearing a total charge of −2. Docking calculations were performed using Autodock 3.0 software43Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function.J Comput Chem. 1998; 19: 1639-1662Crossref Scopus (9639) Google Scholar with default values for all parameters except the number of runs (10), energy evaluations (9.0 × 1010), generations (60,000), and local search iterations (3000). The complex chosen as the best geometry from each docking calculation was that with the lowest final docked energy value. Results are described using a numbering system that facilitates comparisons among homologous positions in G protein–coupled receptors as described by Ballesteros and Weinstein.44Ballesteros J.A. Weinstein H. Chapter 19.in: Conn P.M. Sealfon S.C. Methods in neurosciences. Academic, San Diego1995: 366-428Google Scholar In this system, each amino acid in the transmembrane domain is given a number in the format X.YY where X indicates the number of the transmembrane helix where the amino acid is found and YY indicates the position relative to the most conserved amino acid in that helix at reference position 50. Tritiated OTP was synthesized in our laboratory as shown in Figure 1D. Pyridinium chlorochromate–mediated oxidation of oleyl alcohol gave oleyl aldehyde (2), which was subjected to reduction with NaB3H4 to form the tritiated oleyl alcohol (3). Alcohol 3 was phosphorylated as reported earlier40Durgam G.G. Virag T. Walker M.D. Tsukahara R. Yasuda S. Liliom K. van Meeteren L.A. Moolenaar W.H. Wilke N. Siess W. Tigyi G. Miller D.D. Synthesis, structure-activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of autotaxin.J Med Chem. 2005; 48: 4919-4930Crossref PubMed Scopus (101) Google Scholar to give protected oleyl thiophosphate ester (4). Treatment of 4 with methanolic KOH, followed by acidification, yielded [3H]-OTP. The specific activity of the product was 10.8 mCi/mmol. To determine oral absorption of OTP, female 8- to 10-week-o
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