Effects of calmodulin antagonists on serine phospholipid base-exchange reaction in rabbit platelets.

Effects of the calmodulin antagonists chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide on phospholipid metabolism were examined in rabbit platelets using [3H]serine, [3H]ethanolamine, [3H]choline, and [3H]glycerol. All these drugs markedly stimulated the incorporation of [3H]serine into phosphatidylserine. On the other hand, these drugs had only a slight effect on the rate of incorporation of [3H]ethanolamine and [3H]choline into the corresponding phospholipid. When [3H]glycerol was used as a precursor of the phospholipids, 3H-labeled phospholipids were mainly composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Although the phosphorus content of phosphatidylserine was about 40% of that of phosphatidylcholine in rabbit platelets, the amount of phosphatidylserine labeled with [3H]glycerol was less than 2% of that of the labeled phosphatidylcholine, and calmodulin antagonists slightly stimulated the incorporation of [3H]glycerol into phosphatidylserine. Treatment with calmodulin antagonists caused a marked decrease in the content of endogenous free serine with concomitant increase in the contents of endogenous free ethanolamine and choline. On the other hand, the contents of other free amino acids, including essential and non-essential amino acids, were unchanged. These results suggest that the calmodulin antagonists we used did not affect de novo synthesis of phosphatidylserine, but did stimulate the serine phospholipid base-exchange reaction in rabbit platelets.

Elevated erythrocyte CDP-choline levels associated with beta-thalassaemia in patients with transfusion independent anaemia.

The accumulation of CDP-ethanolamine as well as CDP-choline in a small cohort of patients with normal UMPH1 and no defined cause for their anaemia suggested a defect in both phosphotransferases. Here we report 10 patients with transfusion independent beta-thalassaemia; 8 being pure heterozygotes and 2 heterozygotes also for Hb E. Mean CDP-choline (86.xxx +/- 48 microM) and CDP-ethanolamine (34.6 microM +/- 34.5 microM), mean control <3 microM. Elevated CDP-choline in patients with no defined cause for their haemolytic anaemia was previously suggested as a possible indicator of CDP-choline phosphotransferase deficiency. Here we associate it with transfusion independent beta-thalassaemia.

Distribution of erythrocyte nucleotides in pyrimidine 5'-nucleotidase deficiency.

In pyrimidine 5'-nucleotidase deficiency, erythrocytes contain elevated levels of pyrimidine nucleotides. The composition of this nucleotide pool was examined by ion exchange chromatography on Dowex formate columns using a linear ammonium formate elution gradient. In contradistinction to normal erythrocytes, adenine nucleotides accounted for only 32% of the nucleotide pool. The remainder consisted of 50% cytidine and 16% uridine nucleotides. The remaining 2% was not identified. The most abundant compound appeared to be UDP glucose whilst high levels of CTP, CMP and an unidentified cytidine compound less polar than CMP accounted for most of the cytidine nucleotide pool. The possibility that the abnormal nucleotides were due to an elevated reticulocyte count was excluded and it was also shown that erythrocytes from subjects heterozygous for pyrimidine 5'-nucleotidase deficiency did not have detectable levels of the abnormal nucleotides.

Abnormal erythrocyte pyrimidine nucleotides in uremic subjects.

Uremia causes major increases in the erythrocyte (RBC) purine nucleotides, presumably secondary to phosphate retention, but no previous study has been made of the pyrimidine nucleotides, normally absent from RBC. This investigation was prompted by demonstration of the abnormal presence of RBC pyrimidine nucleotides, primarily cytidine triphosphate (CTP) plus cytidine diphosphate-choline (CDP-C) and cytidine diphosphate-ethanolamine (CDP-E), in two types of congenital hemolytic anemia as well as in lead poisoning. These observations suggested an analogy to the RBC membrane dysfunction of uremia. This is a report of the identification of CDP-C and CDP-E as the predominant abnormal pyrimidine nucleotides in the RBC hemolysates of uremic subjects. High-performance liquid chromatography of hemolysates from uremic adults showed a 50% increase in purine nucleotides and the abnormal presence of pyrimidine nucleotides and diesters at approximately 10% of the concentration of the purine nucleotides. By means of UV spectra and 31P nuclear magnetic resonance, these were identified as CDP-C and CDP-E. The increased purine and abnormal pyrimidine nucleotides of uremic RBC were unrelated to the pre- or posthemodialysis state, allopurinol, levels of blood lead, copper and zinc, or RBC pyrimidine 5'-nucleotidase, the cytosolic enzyme that specifically dephosphorylates the pyrimidine nucleotides. Although the accumulation of CTP, CDP-C and CDP-E may be an epiphenomenon of phosphate retention, it also suggests a common pathway to the accelerated hemolysis of chronic renal insufficiency.

Identification of cytidine diphosphodiesters in erythrocytes from a patient with pyrimidine nucleotidase deficiency.

Pyrimidine 5'-nucleotidase deficient (PND) erythrocytes contain elevated levels of pyrimidine nucleotides and relatively normal purine nucleotide levels. The composition of this nucleotide pool has been examined by others, but not all of the abnormal red cell metabolites in this disorder were identified. We have isolated and positively confirmed the identity of cytidine diphosphate (CDP)-choline and CDP-ethanolamine from PND red cells using methods including proton FT-NMR, spectroscopy, and comparative mass spectrometry. The concentrations of these and other pyrimidine nucleotidase-deficient erythrocyte nucleotides were determined using anion-exchange high performance liquid chromatography and ultraviolet (u.v.) detection. The pyrimidine diphosphodiesters appear to be the most prominent abnormal pyrimidine nucleotides in PND red cells, accounting for 55% of the total red cell pyrimidine nucleotides in this disorder. It is proposed that these abnormal phosphodiesters may be related to the accelerated hemolysis in PND.