Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update

Both a 25-hydroxylation and a 1alpha-hydroxylation are necessary for the conversion of vitamin D(3) into the calcium-regulating hormone 1alpha,25-dihydroxyvitamin D(3). According to current knowledge, the hepatic mitochondrial cytochrome P450 (CYP) 27A and microsomal CYP2D25 are able to catalyze the former bioactivation step. Substantial 25-hydroxylase activity has also been demonstrated in kidney. This paper describes the molecular cloning and characterization of a microsomal vitamin D(3) 25- and 1alpha-hydroxylase in kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D(3) and 1alpha-hydroxyvitamin D(3) and, in addition, 1alpha-hydroxylation of 25-hydroxyvitamin D(3). The cDNA encodes a protein of 500 amino acids. Both the DNA sequence and the deduced peptide sequence of the renal enzyme are homologous with those of the hepatic vitamin D(3) 25-hydroxylase CYP2D25. Genomic Southern blot analysis suggested the presence of a single gene for CYP2D25 in the pig. Immunohistochemistry experiments indicated that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. These findings indicate a tissue-specific developmental regulation of CYP2D25. The results from the current and previous studies on renal vitamin D hydroxylations imply that CYP2D25 has a biological role in kidney.     

Problems of uridine protection

Various routes to synthesize 5'-O-dimethoxytrityl-O4-p-nitrophenylethyl- 2'-O-p-nitrophenylethylsulfonyluridine (7) as a useful intermediate in oligoribonucleotide synthesis have been investigated. The direct sulfonylation of 5'-dimethoxytrityl-O4-p-nitrophenylethyluridine (4) gave the best results despite the fact that 7 is formed in almost equal amounts with its 3'-NPES isomer (8) and the 2',3'-di-O-NPES derivative (6).     

Abnormalities in uridine homeostatic regulation and pyrimidine nucleotide metabolism as a consequence of the deletion of the uridine phosphorylase gene

We report in the present study the critical role of uridine phosphorylase (UPase) in uridine homeostatic regulation and pyrimidine nucleotide metabolism, employing newly developed UPase-/- mice. Our data demonstrate that the abrogation of UPase activity led to greater than a 6-fold increase in uridine concentrations in plasma, a 5-6-fold increase in lung and gut, and a 2-3-fold increase in liver and kidney, as compared with wild type mice. Urine uridine levels increased 24-fold normal in UPase-/- mice. Uridine half-life and the plasma retention of pharmacological doses of uridine were significantly prolonged. Further, in these UPase-/- mice, abnormal uridine metabolism led to disorders of various nucleotide metabolisms. In the liver, gut, kidney, and lung of UPase-/- mice, total uridine ribonucleotide concentrations increased 2-3 times as compared with control mice. Cytidine ribonucleotides and adenosine and guanosine ribonucleotides also increased, although to a lesser extent, in these organs. Most significant deoxyribonucleotide changes were present in the gut and lung of UPase-/- mice. In these tissues, dTTP concentration increased more than 4-fold normal, and dCTP, dGTP, and dATP concentrations rose 1-2 times normal. In kidney, dTTP concentration increased 2-fold normal, and dCTP and dGTP concentrations rose less than 1-fold normal. In addition, the accumulated uridine in plasma and tissues efficiently reduced 5-fluorouracil host toxicity and altered the anesthetic effect of pentobarbital. These data indicate that UPase is a critical enzyme in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism, and 5-fluorouracil activity.     

Multiple controls on the intracellular trapping of uridine

Uridine uptake by mouse or hamster cells grown in conditions which support good growth is very sensitive to inhibition by cyanide and azide, at concentrations which only slightly reduce overall cellular ATP levels. Iodoacetate, when present alone, reduces uridine uptake only insofar as it reduces cellular ATP levels. At concentrations which by themselves do not affect uridine uptake, iodoacetate greatly reduces the sensitivity of uridine uptake to cyanide or azide. The effect of cyanide is on intracellular trapping of uridine and not on its transport into the cell. The specific effect of cyanide is confined to uridine and not found for the uptake of adenine, thymidine or 2-deoxyglucose. The effect is of rapid onset (within 2 min) and is rapidly reversible (also within 2 min). Phosphorylation of uridine in homogenised cells or in Triton X-100-permeabilised cells is unaffected by cyanide. The data are interpreted in terms of a model in which intracellular trapping of uridine is subject to multiple controls, including one regulated by some factor requiring intact functioning of the mitochondrion. These multiple control systems interact synergistically to affect trapping of uridine by the intact cell.     

Incorporation of uridine by the perfused rabbit heart

Uridine is rapidly incorporated into the free pyrimidine nucleotides of the isolated perfused rabbit heart. The initial uptake depends on the concentration of precursor, following a Menten-Michaelis like pattern (apparent Km 5 μM).In a dose of 20 μmole.l⁻¹, amounts of labelled uridine corresponding to about a third of the pool of uracil nucleotides are incorporated during the first half hour of administration. Then the rate or uridine uptake decreases with time while the uracil nucleotide pool size increases.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis

Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.