guanosine-triphosphate and sphingosine-phosphorylcholine

We investigated mechanisms for inhibition of B16 melanoma cell migration and invasion by sphingosine-1-phosphate (S1P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S1P, dihydro-S1P, and sphingosylphosphorylcholine inhibited B16 cell migration and invasion with the relative potencies expected as S1P2 receptor agonists. The S1P2-selective antagonist JTE013 completely abolished the responses to these agonists. In addition, JTE013 abrogated the inhibition by sphingosine, which is the S1P precursor but not an agonist for S1P receptors, indicating that the sphingosine effects were mediated via S1P2 stimulation, most likely by S1P that was converted from sphingosine. S1P induced inhibition and activation, respectively, of Rac and RhoA in B16 cells, which were abrogated by JTE013. Adenovirus-mediated expression of N17Rac mimicked S1P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S1P inhibition. Overexpression of S1P2 sensitized, and that of either S1P1 or S1P3 desensitized, B16 cells to S1P inhibition of Rac and migration. In JTE013-pretreated, S1P3-overexpressing B16 cells, S1P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S1P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE013, S1P instead stimulated Rac and migration in B16 cells that overexpress either S1P1 or S1P3, unveiling counteractions between S1P2 and S1P1 or S1P3 chemotactic receptor.

Topics: Adenoviridae; Animals; Blotting, Northern; Blotting, Western; Calcium; Cell Movement; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genetic Vectors; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Ligands; Lysophospholipids; Melanoma, Experimental; Mice; Neoplasm Invasiveness; Phosphorylcholine; Plasmids; Protein Isoforms; Pyrazoles; Pyridines; rac GTP-Binding Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Temperature; Time Factors

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Cations, Divalent; Cattle; Cells, Cultured; Cysteine; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Guanosine Triphosphate; Guinea Pigs; Humans; Kinetics; Mercaptoethanol; Nitric Oxide; Nitroso Compounds; Phosphorylcholine; Platelet Aggregation; Protein Conformation; Protein Glutamine gamma Glutamyltransferase 2; Recombinant Proteins; S-Nitrosothiols; Sphingosine; Transglutaminases

Sphingosylphosphorylcholine (SPC), a sphingolipid, concentration-dependently (1-50 microM) induced contraction and slight elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle of the pig coronary artery, the result being a marked increase in the force/[Ca(2+)](i) ratio. In alpha-toxin- or beta-escin-permeabilized, but not Triton X-100-permeabilized, vascular strips, SPC induced contraction at constant [Ca(2+)](i) (pCa 6.3) in the absence of GTP, whereas a G-protein-coupled receptor agonist, histamine, required the presence of GTP to induce the contraction. The Rho-kinase blocker, Y-27632 (10 microM) abolished the SPC-induced Ca(2+)-sensitization, without affecting the Ca(2+)-induced contraction. These results suggest that SPC induces Ca(2+)-sensitization of force in vascular smooth muscle, presumably through the activation of Rho-kinase (or a related kinase).

Topics: Amides; Animals; Calcium; Coronary Vessels; Cytosol; Enzyme Inhibitors; Guanosine Triphosphate; Histamine; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Kinetics; Muscle Contraction; Muscle, Smooth, Vascular; Phosphorylcholine; Potassium; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases; Sphingosine; Swine

Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both mu- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.

Topics: Animals; Baculoviridae; Calcium; Calpain; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Guanosine Triphosphate; Humans; Hydrolysis; Nitric Oxide; Phosphorylcholine; Recombinant Proteins; Sphingosine; Spodoptera; Transglutaminases

Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase reaction resulting in the formation of gamma-glutamyl-epsilon-lysine bonds and is activated during apoptosis to catalyze the formation of apoptotic body. We investigate whether lipids that are membrane components and involved in cell signaling could modify the Ca2+-dependent activation of tTG. We found that sphingosylphosphocholine (lyso-SM) was the only lipid to activate transglutaminase at low Ca2+ concentrations. In the presence of lyso-SM (125 microM), transglutaminase was detectable at 10 microM Ca2+, whereas in the absence of lyso-SM, similar activity was obtained at 160 microM Ca2+. Furthermore, in the presence of lipid vesicles lyso-SM retained the ability to enhance the Ca2+-dependent activation of tTG. Lyso-SM did not significantly change the Km for the glutamyl and primary amine substrates. However, the Kact for Ca2+ was reduced from 300 microM to 90 microM. Structure-function studies of lyso-SM analogs indicate that phosphocholine group on C1, the free amino group at C2 and a C4-C5 double bond are critical for the activation of transglutaminase activity. This is the first demonstration that a specific sphingolipid could enhance the activity of tTG and could play a role in vivo in activation of the tTG at physiologic Ca2+ levels.

Topics: Calcium; Enzyme Activation; Factor XIII; Guanosine Triphosphate; Humans; Phosphorylcholine; Sphingosine; Transglutaminases; Trypsin

The effect of sphingosine on the cytosolic free Ca2+ concentrations, [Ca2+]i, of human neutrophils was re-examined using Fura-2 loaded cells. We found that sphingosine induced a dose-dependent elevation of [Ca2+]i. At sphingosine concentrations > or = 10 microM, the rise in [Ca2+]i was biphasic; an initial phase increasing basal [Ca2+]i by 100% was succeeded by a second phase which raised [Ca2+]i to several microM. The enhanced signal was sustained and slowly approached the Fmax of Fura-2 over 10 min. Although cytotoxicity assays indicate that Fura-2 leakage contributed to the rise in fluorescence, EGTA, surprisingly, had no effect on the time course of this response. The explanation was that EGTA blocked Fura-2 leakage from and trypan blue uptake by neutrophils. Thus, in the presence of EGTA, biphasic increases in the fluorescent signal can be attributed mainly to release of intracellular Ca2+. Mn2+ quenching studies confirmed that sphingosine mobilized Ca2+ in two distinct phases and promoted the influx of Mn2+. Mn2+ entry, however, was not matched by substantial Ca2+ influx. Sphingosine elevation of [Ca2+]i was insensitive to pertussis toxin treatment of neutrophils and was not correlated with (1,4,5)IP3 formation. Studies with semi-permeabilized cells show that sphingosine, up to 80 microM, neither mobilized Ca2+ significantly nor inhibited active Ca2+ sequestration. Sphingosylphosphorylcholine induced a small but dose-dependent release of Ca2+. We hypothesize that a metabolite of sphingosine may release Ca2+ directly in intact neutrophils.

Topics: Biological Transport; Calcium; Cell Compartmentation; Cell Death; Dose-Response Relationship, Drug; Edetic Acid; Fluorescent Dyes; Fura-2; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Ionomycin; Manganese; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pertussis Toxin; Phosphorylcholine; Saponins; Sphingosine; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella