In contrast, Ca2+ levels of 0

In contrast, Ca2+ levels of 0.1mM caused inhibition of PAF-CPT in the absence of EDTA ( .05), which Paeoniflorin was partially reduced by the presence of the chelate reagent. Table 2 Effect of Mg2+ cation on PAF-CPT activity of HMC. to 1-pathway the final step includes an acetylation of l-alkyl-2-lyso-pathway should mainly contribute to PAF synthesis for maintaining its basal levels under physiological conditions, whereas the pathway should be more involved in the production Paeoniflorin of PAF during inflammatory responses [7, 8]. However, the information collected so far concerning PAF biosynthetic pathways suggest that the contribution of the aforementioned enzymes to PAF synthesis depends on several factors under physiological and pathological conditions [8C13], and so the above point of view should be reevaluated and further studied. The important regulatory enzyme of the route, PAF-CPT, is widely distributed among mammalian tissues and is located on the cytoplasmic surface of the endoplasmic reticulum [8]. It has been found in a variety of rat tissues [7, 8, 12, 14] with the spleen, lung, liver, and kidney exhibiting the highest activities. Human renal cell carcinoma [13], porcine spleen [11], as well as human neutrophils, human cerebrum, fetal rabbit lungs, and unfertilized mouse oocytes, zygotes, and preimplantation embryos [8, 15] also contain significant amounts of PAF-CPT. PAF-CPT has been solubilized from porcine spleen microsomes using digitonin [11]. Although the activity of the solubilized enzyme was relatively stable, further purification by sequential chromatography caused a remarkable decrease in enzyme activity, which was partially recovered by the exogenously addition of phospholipids such as egg phosphatidylcholine, and so forth. [11]. In contrast, dioleoylphosphatidic acid (DOPA) and lysophospholipids showed an inhibitory effect on enzyme activity [11]. The molecular Paeoniflorin weight of the enzyme solubilized from porcine spleen microsomes Nog was estimated to be 440 kd based on gel-filtration column chromatography, suggesting that this enzyme formed a complex with other protein molecules and membrane phospholipids, and that these phospholipids were necessary to maintain the enzyme activity [11]. Although PAF-CPT and the cholinephosphotransferase involved in phosphatidylcholine synthesis (PC-CPT) have several common features, however significant differences between the two enzymes concerning their behavior to detergents, DTT, ethanol, pH [8], as well as interactions with environmental membrane phospholipids containing phosphatidic acid (PA) and/or lysophospholipids [11] have been observed. All the above data support the hypothesis that PAF-CPT is a separate enzyme from PC-CPT, although further studies are required. Investigation of PAF-CPT substrate specificity of several alkylacetylglycerol substrates has shown that the enzyme prefers alkyl substrates possessing either an acetyl or propionyl group at the and remodeling biosynthetic routes can produce PAF either by intrinsic glomerular cells such as mesangial cells [16] or by infiltrating inflammatory cells. Apart from PAF physiological effects, its increased levels in kidney are involved in the pathogenesis and progression of renal damage [17C19]. The study of PAF metabolic enzymes in kidney, especially in mesangial cells, is of great importance since they regulate PAF levels both intracellularly and extracellularly. In our previous studies, PAF-AH as well as remodeling and acetyltransferases have been previously characterized in cortex and medulla from human kidney tissue [20C22], while remodeling PAF acetyltransferases have been characterized in human mesangial cells [23]. Although PAF metabolism has been described in mesangial cells [24, 25], as far as we know there are no direct studies on PAF-CPT in mesangial cells. The aim of the present work was a biochemical characterization of PAF-PCT in mesangial cells. Moreover, the effects of several bioactive compounds of Mediterranean diet and various drugs on PAF-CPT activity were tested in order to evaluate a possible beneficial effect of these factors on renal disorders. 2. MATERIALS AND METHODS 2.1. Materials and instrumentation Centrifugations were performed in a Heraeus Labofug 400R and a Sorvall RC-5B refrigerated.