Antonio Gómez-Muñoz

Interaction of Ceramides, Sphingosine, and Sphingosine 1-Phosphate in Regulating DNA Synthesis and Phospholipase D Activity

C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C2-ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C2- and C6-ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C2-ceramide on this activation. Therefore, this effect of C2-ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C2-ceramide stimulated the conversion of exogenous [3H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C2- and C6-ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

Phosphatidate Phosphohydrolase Catalyzes the Hydrolysis of Ceramide 1-Phosphate, Lysophosphatidate, and Sphingosine 1-Phosphate*

A Mg2+-independent phosphatidate phosphohydrolase was purified from rat liver plasma membranes in two distinct forms, an anionic protein and a cationic protein. Both forms of the enzyme dephosphorylated phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. When assayed at a constant molar ratio of lipid to Triton X-100 of 1:500, the apparent Km values of the anionic phosphohydrolase for the lipid substrates was 3.5, 1.9, 0.4, and 4.0 μM, respectively. The relative catalytic efficiency of the enzyme for phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate was 0.16, 0.14, 0.48, and 0.04 liter (min·mg)−1, respectively. The hydrolysis of phosphatidate was inhibited competitively by ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. The Ki(app) values were 5.5, 5.9, and 4.0 μM, respectively. The hydrolysis of phosphatidate by the phosphohydrolase conformed to a surface dilution kinetic model. It is concluded that the enzyme is a lipid phosphomonoesterase that could modify the balance of phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate relative to diacylglycerol, ceramide, monoacylglycerol, and sphingosine, respectively. The enzyme could thus play an important role in regulating cell activation and signal transduction.

Effects of sphingosine, albumin and unsaturated fatty acids on the activation and translocation of phosphatidate phosphohydrolases in rat hepatocytes

The activities of two phosphatidate phosphohydrolases were measured in cultured rat hepatocytes incubated with 0.1 mM albumin. The activity, which is inhibited by N-ethylmaleimide (PAP-1) is located in the cytosolic and membrane fractions. PAP-1 activity is stimulated by Mg2+ and it can be translocated from the cytosol to the membranes by relatively low (0.5-1 mM) concentrations of fatty acids. In addition, higher concentrations (1-3 mM) of fatty acids cause an increase in the total PAP-1 activity. Translocation of PAP-1 activity in the hepatocytes is preferentially promoted by unsaturated fatty acids (C18:1, C18:2, C18:3, C20:4 and C20:5), rather than by saturated acids (C14:0, C16:0, C18:0). Increasing the extracellular concentration of albumin from 30 microM to 1 mM displaces PAP-1 activity from the membrane fraction. Sphingosine, but not staurosporine, can inhibit the redistribution of PAP-1 activity induced by oleate. The amphiphilic amines, sphingosine, chlorpromazine and propranolol, also decrease membrane-bound PAP-1 activity in the absence of fatty acids, but they do not alter, significantly, the activity of the cytosolic PAP-1. In the presence of 1 mM oleate, sphingosine, chlorpromazine and propranolol decrease the translocation of PAP-1 from the cytosol to the membranes. The phosphohydrolase activity, which is insensitive to N-ethylmaleimide (PAP-2), is specifically located in the plasma membrane (Jamal, Z., Martin, A., Gomez-Muñoz, A. and Brindley, D.N. (1991) J. Biol. Chem. 266, 2988-2996) and it is not stimulated by Mg2+. Saturated fatty acids, albumin, sphingosine and propranolol have no significant effects on PAP-2 activity. However, chlorpromazine decreases PAP-2 activity by about 14%. Linolenate, arachidonate and eicosapentaenoate at 1 mM also produced small (7-10%) decreases in PAP-2 activity. It is proposed that both PAP-1 and PAP-2 activities may be involved in signal transduction, although the main function of PAP-1 seems to be involved in the synthesis of glycerolipids.

Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes

Incubation of hepatocytes with okadaic acid displaced the N‐ethylmaleimide‐sensitive phosphatidate phosphohydrolase from the membrane fraction into the cytosol and partially prevented the oleate‐induced movement of phosphohydrolase from cytosol to membranes. However, higher concentrations of oleate still caused translocation and activation of the phosphohydrolase. This enzyme is stimulated by Mg2+, and is probably involved in glycerolipid synthesis. Okadaic acid also decreased the concentration of diacylglycerol within the hepatocytes. Okadaic acid had no observable effect on the activity of an N‐ethylmaleimide‐insensitive phosphatidate phosphohydrolase which remained firmly attached to membranes. This activity is not stimulated by Mg2+ and is probably involved in signal transduction by the phospholipase D pathway.

Cholesterol feeding induces hypertriglyceridaemia in hamsters and increases the activity of the Mg2+-dependent phosphatidate phosphohydrolase in the liver

(1) Feeding increased cholesterol to hamsters resulted in a dose-dependent increase in cholesterol and triacylglycerol in very-low-density lipoprotein (VLDL), in serum non-esterified fatty acids and in the activity of the Mg(2+)-dependent phosphatidate phosphohydrolase in liver. (2) The effects of increasing dietary cholesterol by 0.12% (w/w) in addition to feeding fat (14%, w/w) were dependent upon the nature of the fat. Lard in the presence of 0.12% (w/w) cholesterol increased serum triacylglycerols as did olive oil. By contrast, sunflower oil did not cause a significant change in serum triacylglycerol concentrations. (3) There was a highly positive correlation between VLDL triacylglycerol and VLDL cholesterol concentrations suggesting that, at least in this model, there is a close relationship between hypertriglyceridaemia and hypercholesterolaemia.