A more complete understanding of the physiological and pathological role of lysophosphatidic acid (LPA) requires receptor subtype-specific agonists and antagonists. Here, we report the synthesis and pharmacological characterization of fatty alcohol phosphates (FAP) containing saturated hydrocarbon chains from 4 to 22 carbons in length. Selection of FAP as the lead structure was based on computational modeling as a minimal structure that satisfies the two-point pharmacophore developed earlier for the interaction of LPA with its receptors. Decyl and dodecyl FAPs (FAP-10 and FAP-12) were specific agonists of LPA 2 (EC 50 ϭ 3.7 Ϯ 0.2 M and 700 Ϯ 22 nM, respectively), yet selective antagonists of LPA 3 (K i ϭ 90 nM for FAP-12) and FAP-12 was a weak antagonist of LPA 1 . Neither LPA 1 nor LPA 3 receptors were activated by FAPs; in contrast, LPA 2 was activated by FAPs with carbon chains between 10 and 14. Computational modeling was used to evaluate the interaction between individual FAPs (8 to 18) with LPA 2 by docking each compound in the LPA binding site. FAP-12 displayed the lowest docked energy, consistent with its lower observed EC 50 . The inhibitory effect of FAP showed a strong hydrocarbon chain length dependence with C12 being optimum in the Xenopus laevis oocytes and in LPA 3 -expressing RH7777 cells. FAP-12 did not activate or interfere with several other G-protein-coupled receptors, including S1P-induced responses through S1P 1,2,3,5 receptors. These data suggest that FAPs are ligands of LPA receptors and that FAP-10 and FAP-12 are the first receptor subtype-specific agonists for LPA 2 .

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Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA 1-3 . We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA 1-3 predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E max ) and potency (EC 50 ) of LPA-elicited Ca 2؉ transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA 1-3 and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA 1 and LPA 2 more than in LPA 3 . The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA 1 and LPA 2 but reduced the activation and modeled interactions of LPA 3 . The R5.38A mutant of LPA 2 and R5.38N mutant of LPA 3 showed diminished activation by LPA; however, in LPA 1 the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA 2 , K7.36A decreased the potency of LPA; in LPA 1 this same mutation increased the E max . In LPA 3 , R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC 50 in response to LPA 10-fold. In LPA 1-3 , the mutation Q3.29E caused a modest increase in EC 50 in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA 2 and LPA 3 receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors. Lysophosphatidic acid (LPA)2 and sphingosine 1-phosphate (S1P) are structurally related lysophospholipid growth factors that mediate a variety of cellular effects, including regulation of cellular proliferation, survival, migration, and morphology (1-3). LPA has been shown to play an important role in a variety of diseases including ovarian cancer, prostate cancer, breast cancer, and cardiovascular disease (4 -14). Many of the biological effects of LPA are mediated through cell surface receptors of the endothelial differentiation gene (EDG) family of G protein-coupled receptors (GPCRs).The EDG family of GPCRs includes eight closely related genes that show the conserved GPCR topology of an extracellular amino terminus followed by seven ␣-helical transmembrane domains (TMs) (15). Three of these genes (LPA 1-3 ) are cellular receptors for LPA and share 55% overall homology in humans. The other five (S1P 1-5 ) are cellular receptors for S1P and share 50% homology in humans. The two subclusters are 35% homologous with each other. The transmembrane domains of human LPA 1-3 where ligand binding takes place show 81% homology with each other. LPA has also been shown to elicit cellular responses through binding to three non-EDG family GPCRs, p2y9/LPA 4 , GPR92/LPA 5 , and GPR87/LPA 6 , which are more closely related to the purinoreceptor clus...

An "A1 type" phospholipase activity with serine-phospholipid preference was released by rat activated platelets. It was distinct from the secretory type II phospholipase A2 [Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101, 625-631] and co-purified with the secretory lysophosphatidylserine-selective lysophospholipase activity [Higashi, S., Kobayashi, T., Kudo, I., and Inoue, K. (1988) J. Biochem. 103, 442-447]. Several lines of evidence indicated that a single protein was responsible for the phospholipase A1 and lysophospholipase activities. Marked accumulation of lysophospholipids was observed in rat calcium ionophore-activated washed platelets and both phospholipase A1/lysophospholipase and type II phospholipase A2 were shown to contribute to this phospholipid degradation. A selective inhibitor of type II phospholipase A2 reduced the phospholipid degradation and enhanced the clotting time and prothrombinase activity. These results indicate that secretory platelet phospholipases may play a role in regulation of blood clotting.

Excessive nitric oxide (NO) production has been implicated to be responsible for the development of septic shock. To determine whether plasma nitrite/nitrate (NOx) levels are related to the severity of systemic inflammatory response syndrome (SIRS) and the degree of multiple organ dysfunction, we studied plasma NOx levels in 70 patients with SIRS consisting of noninfectious SIRS (n = 32), sepsis (n = 23), and septic shock (n = 15). Infection is a microbial phenomenon characterized by an inflammatory response to the presence of microorganism. Positive culture for microorganism is regarded as infectious SIRS (sepsis and septic shock) and negative culture is regarded as noninfectious SIRS. Plasma samples collected from each patient within 24 h from admission to the intensive care unit were subjected for measurement of NOx levels, the stable end products of NO, by the high performance liquid chromatography-Greiss system. Mean plasma NOx levels in patients with SIRS were 52.8 +/- 44 microM/L, ranging from 8.1 to 186.2 microM/L. Plasma NOx levels were positively correlated with Acute Physiology, Age, and Chronic Health Evaluation (APACHE) III score (r = 0.414, P < 0.01) and sequential organ failure assessment (SOFA) score (r = 0.433, P < 0.01). Plasma NOx levels in patients with sepsis (51.0 +/- 38.5 microM/L) and septic shock (94.5 +/- 53.7 microM/L) were significantly (P < 0.01) higher than those in patients with noninfectious SIRS (25.8 +/- 16.9 microM/L) and healthy subjects (29.6 +/- 8.9 microM/L). Our study shows that plasma NOx levels are increased in patients with infectious, but not noninfectious SIRS, which increase as the severity of SIRS and the development of multiple organ dysfunction syndrome, suggesting its possible pathogenic role in SIRS.