Secondo Dottori

Increased platelet phosphatidylserine exposure and caspase activation in chronic uremia

Summary.  Platelet activation is associated with exposure of the aminophospholipid phosphatidylserine (PS) to the outer hemi‐leaflet of the plasma membrane bilayer, which seems to be involved in the coagulation process. Because platelet activation may occur in patients suffering from chronic uremia, which is frequently associated with a thrombophilic tendency, we studied whether uremic platelets show an increased propensity to expose PS on the outer membrane leaflet and whether this process is linked with important functional and molecular changes. Flow cytometric percentage of annexin V‐positive platelets, a measure of PS externalization, was significantly elevated (P < 0.001) in uremic patients when compared to normal controls under both unstimulated and agonist‐stimulated conditions. Uremic platelet procoagulant activity, as measured by thrombin generation, was more than twice as high (4.13 ± 0.3 µ mL−1) as that found in normal controls (1.86 ± 0.2 µ mL−1). Two independent assays showed that the enzymatic activity of caspase‐3, a protease involved in the loss of membrane PS asymmetry, was significantly greater in the platelets of uremic subjects than in those of healthy controls. PS exposure in agonist‐stimulated platelets was markedly reduced by inhibition of caspase‐3 activity but was not affected by inhibition of calpain activity. These results support the view that the thrombophilic susceptibility of uremic patients may be partly ascribed to increased PS exposure to the outer membrane leaflet of platelets. This process seems to be causally linked to an increase in caspase‐3 activity, particularly during platelet activation.

Acyl-trafficking in membrane phospholipid fatty acid turnover : the transfer of fatty acid from the acyl-L-carnitine pool to membrane phospholipids in intact human erythrocytes

In this work we have investigated the transfer of radioactive palmitic acid between membrane phospholipids and acyl-L-carnitines in intact human erythrocytes. During the incubation period of labeled erythrocyte in non-defatted bovine serum albumin, radioactivity in phosphatidylcholine and phosphatidylethanolamine increased. On the contrary, a decrease of radioactivity in erythrocyte palmitoyl-L-carnitine was observed. 2-Tetradecylglycidic acid, an irreversible erythrocyte carnitine palmitoyltransferase inhibitor, abolished any radioactivity changes in both phospholipids and palmitoyl-L-carnitine. Similar findings were obtained by using erythrocytes labeled with radioactive oleic acid. Our data suggest that in human erythrocytes a carnitine palmitoyltransferase-catalyzed acyl transfer from acyl-L-carnitine to phospholipids, rather than a previously described fatty acid transfer from phosphatidylcholine to phosphatidylethanolamine, is operative.

Effects of l-carnitine and its acetate and propionate esters on the molecular dynamics of human erythrocyte membrane

EPR and fluorescence probes were used in this study to define the effects of L-carnitine and its short-chain esters, acetyl-L-carnitine and propionyl-L-carnitine, on the natural fluidity gradient and molecular packing of phospholipid headgroups of erythrocyte membrane in intact cells. Purified erythrocyte suspensions, labeled with different stearic acid derivatives containing a stable doxyl radical ring at the C-5, C-7, C-12 and C-16, were incubated with 0.5-5 mM L-carnitine and its esters for 60 min at 37 degrees C and washed twice with an isosmotic buffer. A decrease in the order parameter, calculated from the EPR spectra of the 5-doxylstearic acid derivative, was observed at all the concentrations of propionyl-L-carnitine and the extent of the decrease was dose and temperature dependent. An increase of the chain length between the doxyl ring and the carboxylic group of the spin label, resulted in a much lower efficacy of propionyl-L-carnitine in decreasing the order parameter. Acetyl-L-carnitine also showed a significant effect of decreasing the molecular order but only at the lower temperatures of red cells labeled with 5-doxyl and treated with the highest concentration of the drug. L-Carnitine did not modify the molecular dynamics at all the temperatures and concentrations used in this study. L-Carnitine and its short-chain derivatives did not alter significantly membrane fluidity of deeper regions of the erythrocyte membrane, measured by means of the excimer/monomer fluorescence intensity ratio of pyrene incorporated into the membrane of intact erythrocytes. However, these compounds were all capable of loosening the molecular packing of the polar head of erythrocyte membrane phospholipids evaluated by the membrane binding fluorescence properties of merocyanine-540. The binding of the fluorescent probe decreased in the order propionyl-L-carnitine > acetyl-L-carnitine > L-carnitine. Our findings suggest that this category of compounds affect the molecular dynamics of a membrane bilayer region close to the glycerol backbone of phospholipids, which might be relevant for the expression of membrane functions.

l‐carnitine decreases glycolysis in liquid‐stored platelets

BACKGROUND: The platelet storage lesion is characterized metabolically by a pH decrease associated with lactic acid generation; a change in platelet morphology from discoid to spherical; a diminished response to in vitro challenge tests, such as the hypotonic shock response (HSR) and extent of shape change (ESC); increased surface P‐selectin expression; and decreased in vivo recovery and survival. Altering storage conditions to improve these measures could allow for extension of the duration of in vitro storage.

Participation of carnitine palmitoyltransferase in the synthesis of dipalmitoylphosphatidylcholine in rat alveolar type II cells

We have investigated the role of carnitine palmitoyltransferase (EC 2.3.1.21) in pulmonar type II pneumocyte, a lung cell responsible for the synthesis of surface active lipids. Adult type II pneumocytes were isolated from rat lung and purified by differential adherence. When these lung cells were incubated with radioactive palmitate, the percentage of radioactivity recovered into dipalmitoylphosphatidylcholine (DPPC), a major surface active lipid, was almost 60% with respect to total phosphatidylcholine (PC) molecular species. Cellular lysates from type II pneumocytes contained detectable amount of carnitine palmitoyltransferase (CPT) activity (1 nmol/min/mg). Most of the CPT activity found in these cells could be inhibited by incubating them for 60 min with 5 μM tetradecylglycidic acid (TDGA), a specific and irreversible CPT inhibitor of the malonyl-CoA sensitive CPT isoform (CPT I). TDGA treatment of adult type II pneumocytes caused a significant reduction in the incorporation of radioactive palmitate into PC, though this effect did not seem to be specific for DPPC. TDGA affected the incorporation of radioactive palmitate at the sn2 rather than the sn1 position of the glycerol backbone of PC. The incorporation of radioactive palmitate into DPPC was also observed when these lung cells were incubated with palmitate-labeled palmitoyl-L-carnitine. Our data suggest that type II pneumocyte CPT may play an important role in remodelling PC fatty acid composition and hence DPPC synthesis.

Effect of propionyl-L-carnitine treatment on membrane phospholipid fatty acid turnover in diabetic rat erythrocytes

In this work we have examined the effect of the oral administration of propionyl-L-carnitine (PLC) on the membrane phospholipid fatty acid turnover of erythrocytes from streptozotocin-induced diabetic rats. A statistically significant reduction in radioactive palmitate, oleate, and linoleate, but not arachidonate, incorporation into membrane phosphatidylcholine (PC) of diabetic rat erythrocytes with respect to control animals was found. Changes in radioactive fatty acid incorporation were also found in diabetic red cell phosphatidylethanolamine (PE), though they were not statistically significant. Oral propionyl-L-carnitine (PLC) treatment of diabetic rats partially restored the ability of intact red cells to reacylate membrane PC with palmitate and oleate, and reacylation with linoleate was fully restored. The analysis of the membrane phospholipid fatty acid composition revealed a consistent increase of linoleate levels in diabetic rat red cells, and a modest decrease of palmitate, oleate and arachidonate. The phospholipid fatty acid composition of diabetic red blood cells was not affected by the PLC treatment. Lysophosphatidylcholine acyl-CoA transferase (LAT) specific activity measured with either palmitoyl-CoA or oleyl-CoA was significantly reduced in diabetic erythrocyte membranes in comparison to controls. In addition LAT kinetic parameters of diabetic erythrocytes were altered. The reduced LAT activity could be partially corrected by PLC treatment of diabetic rats. Our data suggest that the impaired erythrocyte membrane physiological expression induced by the diabetic disease may be attenuated by the beneficial activity of PLC on the red cell membrane phospholipid fatty acid turnover.

Is the carnitine system part of the heart antioxidant network

Free radical reactions are considered to be an important pathophysiologic determinant of a broad range of inflammatory and ischemic diseases. The latter disease state, in particular, is still a very active area of free radical research since the discovery of the so-called oxygen paradox [1, 2]. Indeed, the sudden reoxygenation of the myocardium after a transient period of global ischemia results in cellular necrosis and intracellular calcium overload. Current hypotheses on the involvement of oxygen-derived free radicals in the course of the reperfusion event are essentially based on animal studies, in which the inclusion of free radical scavengers in the perfusion medium significantly improved hemodynamic and biochemical parameters [3-6]. In addition, electron spin resonance, low level chemiluminescence and reflectance studies on perfused rat heart suggest that free radical production is already present in the ischemic phase and that shortly after the onset of the oxygen readmission, a burst of free radical generation occurs [7–9].

The Carnitine System and Acyl Trafficking in CNS

The carnitine system may be defined as a family of different short and long-chain acyltransferases, translocases, and their related substrates, whose common denominator is L-carnitine. The carnitine system is best known for the role played in mitochondrial metabolism, though the presence of carnitine-dependent short and long-chain acyltransferases in extra-mitochondrial compartments raises a number of intringuing question about their role. We have recently proposed that carnitine palmitoyltransferase (CPT) may be important in the pathway of phospholipid and triglyceride fatty acid turnover in neurons. The CPT action is accomplished by modulating the size and composition of the acyl-CoA pool between the activation step of the fatty acid and its transfer into complex lipids. In addition, studies on the metabolic fate of the acetate moiety of acetyl-L-carnitine revealed that the lipogenic acetyl-CoA pools present in different cellular compartments of rat brain are not necessary homogeneous. Taken together, these data suggest that the carnitine system may influence key regulatory points of lipid biosynthetic pathways.