Nóra Nusser

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.

Different roles for RhoA during neurite initiation, elongation, and regeneration in PC12 cells.

Abstract : The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)‐induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid‐regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites ; however, they remained responsive to NGF, as indicated by the activation of extracellular signal‐regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered ; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were ~50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis ; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF‐induced neuronal differentiation and neurite regeneration.

Molecular basis for lysophosphatidic acid receptor antagonist selectivity

Recent characterization of lysophosphatidic acid (LPA) receptors has made possible studies elucidating the structure-activity relationships (SAR) for agonist activity at individual receptors. Additionally, the availability of these receptors has allowed the identification of antagonists of LPA-induced effects. Two receptor-subtype selective LPA receptor antagonists, one selective for the LPA1/EDG2 receptor (a benzyl-4-oxybenzyl N-acyl ethanolamide phosphate, NAEPA, derivative) and the other selective for the LPA3/EDG7 receptor (diacylglycerol pyrophosphate, DGPP, 8:0), have recently been reported. The receptor SAR for both agonists and antagonists are reviewed, and the molecular basis for the difference between agonism and antagonism as well as for receptor-subtype antagonist selectivity identified by molecular modeling is described. The implications of the newly available receptor-subtype selective antagonists are also discussed.

Pharmacological Characterization of Phospholipid Growth-Factor Receptors

Abstract: The phospholipid growth‐factor (PLGF) 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 PSP24α and different members of the EDG1‐7 receptors in the same cell. Stable heterologous expression of the PSP24α, 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.

Virtual Screening for LPA2-Specific Agonists Identifies a Nonlipid Compound with Antiapoptotic Actions

Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA2 G protein-coupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L5547924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA2 receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro. Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA1&2 double-knockout mice reconstituted with the LPA2 receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA2, Na+-H+ exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA2-specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.

Sphingosylphosphorylcholine is a bona fide mediator regulating heart rate

KÁROLY LILIOM,a,b,f MORITZ BÜNEMANN,c GUOPING SUN,d DUANE MILLER,d DOMINIC M. DESIDERIO,e BODO BRANDTS,c KIRSTEN BENDER,c LUTZ POTT,c NÓRA NUSSER,a AND GÁBOR TIGYIa aDepartment of Physiology, The University of Tennessee Memphis, Tennessee 38163, USA bInstitute of Enzymology, BRC, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary cInstitut für Physiologie, Ruhr-Universität, D-44780 Bochum, Germany dDepartment of Pharmaceutical Sciences, The University of Tennessee Memphis, Tennessee 38163, USA eThe Charles B. Stout Neuroscience Mass Spectroscopy Laboratory, The University of Tennessee Memphis, Tennessee 38163, USA