Lysophosphatidic acid-induced platelet shape change revealed through LPA(1-5) receptor-selective probes and albumin.

Lysophosphatidic acid (LPA), a component of mildly-oxidized LDL and the lipid rich core of atherosclerotic plaques, elicits platelet activation. LPA is the ligand of G protein-coupled receptors (GPCR) of the EDG family (LPA(1-3)) and the newly identified LPA(4-7) subcluster. LPA(4), LPA(5) and LPA(7) increase cellular cAMP levels that would induce platelet inhibition rather than activation. In the present study we quantified the mRNA levels of the LPA(1-7) GPCR in human platelets and found a rank order LPA(4) = LPA(5) > LPA(7) > LPA(6) = LPA(2) >> LPA(1) > LPA(3). We examined platelet shape change using a panel of LPA receptor subtype-selective agonists and antagonists and compared them with their pharmacological profiles obtained in heterologous LPA(1-5) receptor expression systems. Responses to different natural acyl and alkyl species of LPA, and octyl phosphatidic acid analogs, alpha-substituted phosphonate analogs, N-palmitoyl-tyrosine phosphoric acid, N-palmitoyl-serine phosphoric acid were tested. All of these compounds elicited platelet activation and also inhibited LPA-induced platelet shape change after pre-incubation, suggesting that receptor desensitization is likely responsible for the inhibition of this response. Fatty acid free albumin (10 microM) lacking platelet activity completely inhibited platelet shape change induced by LPA with an IC(50) of 1.1 microM but had no effect on the activation of LPA(1,2,3,&5) expressed in endogenously non-LPA-responsive RH7777 cells. However, albumin reduced LPA(4) activation and shifted the dose-response curve to the right. LPA(5) transiently expressed in RH7777 cells showed preference to alkyl-LPA over acyl-LPA that is similar to that in platelets. LPA did not increase cAMP levels in platelets. In conclusion, our results with the pharmacological compounds and albumin demonstrate that LPA does not induce platelet shape change simply through activation of LPA(1-5), and the receptor(s) mediating LPA-induced platelet activation remains elusive.

A single amino acid determines lysophospholipid specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors.

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P(1)/EDG1 and LPA(1)/EDG2, respectively. We have developed a model of LPA(1) that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA(1)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P(1) study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P(1) and LPA(1) validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA(1), producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors.

Short-chain phosphatidates are subtype-selective antagonists of lysophosphatidic acid receptors.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are members of the phospholipid growth factor family. A major limitation in the field to date has been a lack of receptor subtype-specific agonists and antagonists. Here, we report that dioctylglycerol pyrophosphate and dioctylphosphatidic acid are selective antagonists of the LPA(1) and LPA(3) receptors, but prefer LPA(3) by an order of magnitude. Neither molecule had an agonistic or antagonistic effect on LPA(2) receptor. Consistent with this receptor subtype selectivity, dioctylglycerol pyrophosphate inhibited cellular responses to LPA in NIH3T3 fibroblasts, HEY ovarian cancer cells, PC12 pheochromocytoma cells, and Xenopus laevis oocytes. Responses elicited by S1P in these cell lines that endogenously express S1P(1), S1P(2), S1P(3), and S1P(5) receptors were unaffected by dioctylglycerol pyrophosphate. Responses evoked by the G protein-coupled receptor ligands acetylcholine, serotonin, ATP, and thrombin receptor-activating peptide were similarly unaffected, suggesting that the short-chain phosphatidates are receptor subtype-specific lysophosphatidate antagonists.

Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry.

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.

Physiological responses to lysophosphatidic acid and related glycero-phospholipids.

1-Acyl-2-hydroxy(lyso)-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) has attracted a lot of attention in recent years due to the wide range of its biological effects that span the phylogenetic tree from slime mold to human. LPA can be viewed as a pleiotropic phospholipid growth factor that utilizes the same signal transduction mechanisms as traditional polypeptide growth factors; however, LPA activates these mechanism via specific G protein-coupled receptors. The concentration of LPA in serum is in the high micromolar range, making it the most abundant mitogen/survival factor present in serum, one that is often unknowingly utilized in tissue culture. The present review gives a historical perspective and a critical analysis of the LPA literature with a special emphasis on the physiological implications of its effects.

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo.

The lipid mediator lysophosphatidic acid (LPA) regulates cell proliferation and enhances cell motility in vitro, both of which are important events in wound healing. To evaluate the effects of LPA in vivo, it was applied to a full-thickness wound of rat skin. LPA in micromolar concentrations, or solvent, was applied daily. Animals were killed at 1, 3, 6, and 9 days after wounding and processed for histological evaluation, including hematoxylin-eosin staining and histochemical markers for macrophage-histiocytes, proliferating cells, and capillary endothelial cells. LPA treatment accelerated wound closing and increased neoepithelial thickness. Cytological evaluation showed no evidence for a secondary inflammation-mediated injury, infection, or increased keloid formation. Whereas LPA caused only a modest dose-dependent increase in proliferating cells, a marked increase in the immigration of histiocyte-macrophage cells was observed as early as day 1. The peaks of several cytological features and immunohistological markers preceded those of the untreated side. Our data suggest that exogenously applied LPA in this model promotes healing and that macrophage-histiocytes are the primary LPA-responsive cells in vivo.

Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells.

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [(32)P]Sph-1-P, but not [(32)P]LPA, was observed in [(32)P]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca(++) mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate.

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

Lipid phosphate phosphatase-1 and Ca2+ control lysophosphatidate signaling through EDG-2 receptors.

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 degrees C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca(2+), activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K(d) and B(max) for LPA binding at 4 degrees C, increasing Ca(2+) from 0 to 50 micrometer increased the K(d) from 40 to 900 nm. Decreasing extracellular Ca(2+) from 1.8 mm to 10 micrometer increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca(2+) dependence of the apparent K(d) values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca(2+) levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation.

Pharmacological characterization of phospholipid growth-factor receptors.

The phospholipid growth-factor (PLGE) terminology is proposed to describe a group of endogenous glycerol- and sphingolipid mediators that regulate cell proliferation through plasma membrane receptors. In addition to LPA and SPP, multiple PLGFs are present in blood plasma and serum. PLGF activity is regulated by its stimulus-coupled production and by endogenous inhibitors. In addition to LPA and SPP, alkenyl-glycerophosphate, cyclic-phosphatidic acid, and sphingosylphosphorylcholine were detected in biological fluids using mass spectrometry. Heterologous desensitization studies indicate the expression of multiple LPA-activated receptors in a variety of cell types, which are differentially activated by the different PLGFs. Northern blot and RT-PCR results reinforce the coexpression of PSP24 alpha and different members of the EDG1-7 receptors in the same cell. Stable heterologous expression of the PSP24 alpha, EDG2, and EDG4 receptors in HEK293 cells show distinct PLGF specificities and dose-response properties for each receptor subtype. Thus, both the controlled availability of the different agonists/inhibitors and the regulated expression of their receptors regulate the biological effects of PLGFs.

Phospholipid growth factors and corneal wound healing.

In many tissue types, wound healing involves cell division and migration over and into the wound area to cover and remodel the wound. LPA and other members of the phospholipid lipid growth factor (PLGF) family stimulate many of the activities involved in wound healing. In the rabbit cornea, we have found that keratocytes from wounded corneas have a volume-activated Cl- current activated by LPA and alkenyl-LPA. This current is minimally activated by cyclic PA and SPC, and is not activated by LPA in cells from uninjured corneas. Biochemical examination of PLGFs in aqueous humor and lacrimal fluid before and after wounding identified LPA, alkenyl-GP, PA, and lyso PS, with elevated PLGF activity after wounding. In recent experiments examining human corneal cell lines and cultured cells using RT-PCR, we found mRNA for EDG receptors 1-5, with an apparent increase in EDG-3, -4, and -5 following brief SDS application to cell lines, and EDG receptors 2-5 induction in late-passage human corneal epithelial cells. This work points to a significant role for PLGFs in the corneal wound-healing process.