Abnormalities in Uridine Homeostatic Regulation and Pyrimidine Nucleotide Metabolism as a Consequence of the Deletion of the Uridine Phosphorylase Gene

Abstract

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. 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. Uridine phosphorylase (UPase) 1The abbreviations used are: UPase, uridine phosphorylase; UPase–/–, uridine phosphorylase gene knock-out; 5-FU, 5-fluorouracil; RT, reverse transcription; HPLC, high-performance liquid chromatography; LORR, loss of righting reflex.1The abbreviations used are: UPase, uridine phosphorylase; UPase–/–, uridine phosphorylase gene knock-out; 5-FU, 5-fluorouracil; RT, reverse transcription; HPLC, high-performance liquid chromatography; LORR, loss of righting reflex. is an enzyme that catalyzes the phosphorolysis of uridine into uracil. In the presence of ribose-1-phosphate, UPase can also catalyze the reverse reaction, forming uridine in can into uridine of the of of 5-fluorouracil in normal and tissues is an in the pyrimidine and the liver and kidney, pyrimidine and tissues with uridine normal tissues in the of uridine plasma uridine to increase in as compared with to in pyrimidine 2–3-fold In the plasma concentration of uridine is to pyrimidine of that uridine in the led to a increase in the of that with a of pyrimidine that the and more than of the pyrimidine the of plasma uridine in also in the regulation of and In the of uridine can as an metabolism and the to pyrimidine the the phosphorolysis of uridine can the to is used in the of In the uridine as a of and the to metabolism and In the uridine is in the of with and as addition, the levels of uridine in plasma and tissues also critical the of pyrimidine and that can the of uridine of uridine UPase as to and of of these in is that the uridine plasma concentration is the of homeostasis of uridine and the of to an enzyme uridine UPase an in the regulation of uridine In a that the UPase the changes in uridine metabolism, the of uridine homeostasis pyrimidine nucleotide metabolism, and the toxicity of of UPase–/– with normal were into that in were used to the mice. UPase–/– and wild of the were used the of the were and the abrogation of the UPase gene and uridine activity were to the of with in and a of the with and used to the of UPase tissues, and the as were with in and the reverse in activity the of to and uridine levels were the of as of Uridine and of uridine and ribonucleotides were high-performance liquid and were with of and and with an of Uridine a with as a ribonucleotide were the and the a a were and of the of were a were with and were in a of of and dATP and were and were the were with the of the the the were of were in a and the of uridine in UPase–/– mice, of into a and were and in plasma were and of pharmacological doses of of uridine and were with uridine concentrations were as anesthetic of the loss of righting type and UPase–/– were with of pentobarbital. of the the of in the the of and the wild type and UPase–/– were doses of to were as a host doses to loss were as the the of the UPase UPase gene and the UPase were as in UPase–/– were the and of normal of the were wild type UPase as an the as a to the of a UPase a and an demonstrate a and UPase activity indicate that UPase gene were in UPase–/– a uridine activity present in liver tissues and the of uridine to UPase gene also uridine activity. increase in uridine activity in liver and of UPase–/– mice, uridine activity in the lung and gut the of changes in and as compared with wild type UPase a in UPase mice. were also significant present in the liver, gut, and of Uridine in UPase–/– the of UPase gene uridine metabolism, the concentrations of uridine in the plasma and of UPase–/– mice. compared with wild type mice, the uridine concentration increased more than 6-fold in the plasma of UPase–/– mice, and in tissues, the uridine concentration times normal in lung and gut and 2–3 times normal in liver and a critical role UPase in the regulation of uridine concentrations in plasma and In addition, a of uridine concentration a normal in UPase–/– mice, the role of the in uridine homeostatic regulation of UPase concentrations in plasma, and tissues of UPase in a of uridine in UPase–/– were also the of In wild type mice, doses of were with a the the in UPase–/– and an present in wild type plasma in that of UPase–/– in the that the phosphorolysis UPase in wild type mice. the of UPase gene increased the plasma retention of pharmacological doses of In UPase–/– mice, a pharmacological of of uridine plasma uridine concentration more than the plasma uridine in to in wild type with doses of uridine metabolism in UPase–/– mice. half-life of and were to the levels with as and indicate wild of were the of and with present in wild type mice, in UPase–/– mice, the role of UPase in uridine is the and is the uridine of pharmacological doses of of uridine were and were as and indicate of in UPase–/– of uridine in plasma and tissues, as as the of uridine activity in to in pyrimidine nucleotide metabolism in UPase–/– uridine an role in the pyrimidine the of total ribonucleotides and in the liver, gut, and of UPase–/– mice. in total uridine ribonucleotide concentrations increased 2–3 times normal in and guanosine ribonucleotides and also increased in these to a lesser the increased in these UPase–/– tissues with greater and compared with wild type dTTP concentrations in UPase–/– were more than in gut and lung and in dCTP increased in the gut, and of UPase–/– mice. in and were also in gut, and kidney, were less than the increase of deoxyribonucleotide in UPase–/– less than that the liver, a in a Further, to the of nucleotide in various tissues, the of nucleotide metabolism were also with the loss of the normal and metabolism in UPase–/– mice. ribonucleotide concentrations in of were and as and wild type wild knock-out; total uridine total total adenosine total guanosine of ribonucleotide in UPase–/– mice. deoxyribonucleotide concentrations in of were and as and wild type wild of deoxyribonucleotide in UPase–/– host toxicity in UPase–/– mice, of were various and the toxicity to the in of the in wild type mice, loss doses of in the In UPase–/– mice, of and toxicity in UPase–/– with that in wild type mice. to and were to 5-fluorouracil wild of UPase–/– to the of the uridine levels and in UPase–/– mice. in these a significantly reduced anesthetic effect the UPase–/– mice. a of in wild type mice, the of of in UPase–/– the of in UPase–/– compared with wild type anesthetic and mice. UPase–/– wild is an enzyme that the of uridine to uracil. Our and also the of UPase to catalyze the of into with a In UPase–/– mice, the role of UPase in the regulation of uridine homeostasis in and the effect of uridine homeostatic nucleotide metabolism, 5-FU, and These UPase–/– in UPase a that UPase is an gene of the UPase gene in in significant in uridine and pyrimidine nucleotide UPase–/– mice, and UPase activity a abrogation of UPase gene in tissues of uridine activity to the of activity UPase gene and to the presence of in UPase–/– mice. the presence of an to the UPase gene in and as were In is in the kidney, the gene is in the with that a uridine activity of present in the liver of UPase–/– mice, is to In addition, the of uridine in the tissues of UPase–/– the of uridine in the liver and in the gut and UPase–/– mice, the in uridine and pyrimidine nucleotide metabolism were in the of uridine in plasma and tissues and in the of various nucleotide with the loss of the normal pyrimidine and and the a of plasma uridine uridine UPase abrogation also the of as doses of to a 5–6-fold increase in half-life and the of present in wild type mice. in uridine metabolism a of pharmacological doses of In UPase–/– mice, of uridine the plasma uridine concentration to more than more than the plasma uridine concentration in wild type mice. These data indicate an role UPase in uridine metabolism in these UPase–/– that UPase is metabolism in is an of pyrimidine the uridine uridine can to and to pyrimidine and In UPase–/– mice, significant in the concentrations of various pyrimidine total uridine total dCTP, and These changes were present in tissues also although to a lesser extent, an increase in the concentrations of total guanosine total adenosine dGTP, and in the organs. a the changes of the We that to and to the and pyrimidine the of and pyrimidine of the and effect of UPase activity and pyrimidine nucleotide metabolism that UPase a role in the of the uridine and and various and the and in the of is that the of is a in the regulation of these also a to the to increased the of and uridine is an of and pyrimidine normal tissues as and In UPase–/– with plasma and uridine concentrations a significant to 5-FU, the role that uridine in host is that UPase can catalyze the of 5-FU, forming the pyrimidine also to the of the reduced toxicity in UPase–/– mice. the that is to UPase can as a to activity to is a significant in UPase–/– mice, the plasma levels of uridine in these the anesthetic effect of to a and as compared with wild type mice. to the of to the uridine to the the critical role of UPase in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism UPase–/– mice. of pyrimidine metabolism UPase abrogation the and of increased to 5-FU, and reduced to pentobarbital. study the of 5-FU, and and the of the of these Uridine phosphorylase (UPase) 1The abbreviations used are: UPase, uridine phosphorylase; UPase–/–, uridine phosphorylase gene knock-out; 5-FU, 5-fluorouracil; RT, reverse transcription; HPLC, high-performance liquid chromatography; LORR, loss of righting reflex.1The abbreviations used are: UPase, uridine phosphorylase; UPase–/–, uridine phosphorylase gene knock-out; 5-FU, 5-fluorouracil; RT, reverse transcription; HPLC, high-performance liquid chromatography; LORR, loss of righting reflex. is an enzyme that catalyzes the phosphorolysis of uridine into uracil. In the presence of ribose-1-phosphate, UPase can also catalyze the reverse reaction, forming uridine in can into uridine of the of of 5-fluorouracil in normal and tissues Uridine is an in the pyrimidine and the liver and kidney, pyrimidine and tissues with uridine normal tissues in the of uridine plasma uridine to increase in as compared with to in pyrimidine 2–3-fold In the plasma concentration of uridine is to pyrimidine of that uridine in the led to a increase in the of that with a of pyrimidine that the and more than of the pyrimidine the of plasma uridine in Uridine also in the regulation of and In the of uridine can as an metabolism and the to pyrimidine the the phosphorolysis of uridine can the to is used in the of In the uridine as a of and the to metabolism and In the uridine is in the of with and as In addition, the levels of uridine in plasma and tissues also critical the of pyrimidine and that can the of uridine of uridine UPase as to and of of these in is that the uridine plasma concentration is the of homeostasis of uridine and the of to an enzyme uridine UPase an in the regulation of uridine In a that the UPase the changes in uridine metabolism, the of uridine homeostasis pyrimidine nucleotide metabolism, and the toxicity of of UPase–/– with normal were into that in were used to the mice. UPase–/– and wild of the were used the of the were and the abrogation of the UPase gene and uridine activity were to the of with in and a of the with and used to the of UPase tissues, and the as were with in and the reverse in activity the of to and uridine levels were the of as of Uridine and of uridine and ribonucleotides were high-performance liquid and were with of and and with an of Uridine a with as a ribonucleotide were the and the a a were and of the of were a were with and were in a of of and dATP and were and were the were with the of the the the were of were in a and the of uridine in UPase–/– mice, of into a and were and in plasma were and of pharmacological doses of of uridine and were with uridine concentrations were as anesthetic of the loss of righting type and UPase–/– were with of pentobarbital. of the the of in the the of and the wild type and UPase–/– were doses of to were as a host doses to loss were as the the of UPase–/– with normal were into that in were used to the mice. UPase–/– and wild of the were used the of the were and the abrogation of the UPase gene and uridine activity were to the of with in and a of the with and used to the of UPase tissues, and the as were with in and the reverse in activity the of to and uridine levels were the of as of Uridine and of uridine and ribonucleotides were high-performance liquid and were with of and and with an of Uridine a with as a ribonucleotide were the and the a a were and of the of were a were with and were in a of of and dATP and were and were the were with the of the the the were of were in a and Uridine the of uridine in UPase–/– mice, of into a and were and in plasma were and of pharmacological doses of of uridine and were with uridine concentrations were as anesthetic of the loss of righting type and UPase–/– were with of pentobarbital. of the the of in the the of and the wild type and UPase–/– were doses of to were as a host doses to loss were as the the of the UPase UPase gene and the UPase were as in UPase–/– were the and of normal of the were wild type UPase as an the as a to the of a UPase a and an demonstrate a and UPase activity indicate that UPase gene were in UPase–/– a uridine activity present in liver tissues and the of uridine to UPase gene also uridine activity. increase in uridine activity in liver and of UPase–/– mice, uridine activity in the lung and gut the of changes in and as compared with wild type UPase a in UPase mice. were also significant present in the liver, gut, and of Uridine in UPase–/– the of UPase gene uridine metabolism, the concentrations of uridine in the plasma and of UPase–/– mice. compared with wild type mice, the uridine concentration increased more than 6-fold in the plasma of UPase–/– mice, and in tissues, the uridine concentration times normal in lung and gut and 2–3 times normal in liver and a critical role UPase in the regulation of uridine concentrations in plasma and In addition, a of uridine concentration a normal in UPase–/– mice, the role of the in uridine homeostatic regulation of UPase concentrations in plasma, and tissues of UPase in a of uridine in UPase–/– were also the of In wild type mice, doses of were with a the the in UPase–/– and an present in wild type plasma in that of UPase–/– in the that the phosphorolysis UPase in wild type mice. the of UPase gene increased the plasma retention of pharmacological doses of In UPase–/– mice, a pharmacological of of uridine plasma uridine concentration more than the plasma uridine in to in wild type with doses of uridine of in UPase–/– of uridine in plasma and tissues, as as the of uridine activity in to in pyrimidine nucleotide metabolism in UPase–/– uridine an role in the pyrimidine the of total ribonucleotides and in the liver, gut, and of UPase–/– mice. in total uridine ribonucleotide concentrations increased 2–3 times normal in and guanosine ribonucleotides and also increased in these to a lesser the increased in these UPase–/– tissues with greater and compared with wild type dTTP concentrations in UPase–/– were more than in gut and lung and in dCTP increased in the gut, and of UPase–/– mice. in and were also in gut, and kidney, were less than the increase of deoxyribonucleotide in UPase–/– less than that the liver, a in a Further, to the of nucleotide in various tissues, the of nucleotide metabolism were also with the loss of the normal and metabolism in UPase–/– mice. ribonucleotide concentrations in of were and as and wild type wild knock-out; total uridine total total adenosine total guanosine of ribonucleotide in UPase–/– mice. deoxyribonucleotide concentrations in of were and as and wild type wild of deoxyribonucleotide in UPase–/– host toxicity in UPase–/– mice, of were various and the toxicity to the in of the in wild type mice, loss doses of in the In UPase–/– mice, of and toxicity in UPase–/– with that in wild type mice. to and were to 5-fluorouracil wild of UPase–/– to the of the uridine levels and in UPase–/– mice. in these a significantly reduced anesthetic effect the UPase–/– mice. a of in wild type mice, the of of in UPase–/– the of in UPase–/– compared with wild type anesthetic and mice. UPase–/– wild of the UPase UPase gene and the UPase were as in UPase–/– were the and of normal of the were wild type UPase as an the as a to the of a UPase a and an demonstrate a and UPase activity indicate that UPase gene were in UPase–/– a uridine activity present in liver tissues and the of uridine to UPase gene also uridine activity. increase in uridine activity in liver and of UPase–/– mice, uridine activity in the lung and gut UPase–/– the of changes in and as compared with wild type UPase a in UPase mice. were also significant present in the liver, gut, and of Uridine in UPase–/– the of UPase gene uridine metabolism, the concentrations of uridine in the plasma and of UPase–/– mice. compared with wild type mice, the uridine concentration increased more than 6-fold in the plasma of UPase–/– mice, and in tissues, the uridine concentration times normal in lung and gut and 2–3 times normal in liver and a critical role UPase in the regulation of uridine concentrations in plasma and In addition, a of uridine concentration a normal in UPase–/– mice, the role of the in uridine homeostatic regulation of UPase mice. of uridine in UPase–/– were also the of In wild type mice, doses of were with a the the in UPase–/– and an present in wild type plasma in that of UPase–/– in the that the phosphorolysis UPase in wild type mice. the of UPase gene increased the plasma retention of pharmacological doses of In UPase–/– mice, a pharmacological of of uridine plasma uridine concentration more than the plasma uridine in to in wild type with doses of uridine of in UPase–/– of uridine in plasma and tissues, as as the of uridine activity in to in pyrimidine nucleotide metabolism in UPase–/– uridine an role in the pyrimidine the of total ribonucleotides and in the liver, gut, and of UPase–/– mice. in total uridine ribonucleotide concentrations increased 2–3 times normal in and guanosine ribonucleotides and also increased in these to a lesser the increased in these UPase–/– tissues with greater and compared with wild type dTTP concentrations in UPase–/– were more than in gut and lung and in dCTP increased in the gut, and of UPase–/– mice. in and were also in gut, and kidney, were less than the increase of deoxyribonucleotide in UPase–/– less than that the liver, a in a Further, to the of nucleotide in various tissues, the of nucleotide metabolism were also with the loss of the normal and in UPase–/– host toxicity in UPase–/– mice, of were various and the toxicity to the in of the in wild type mice, loss doses of in the In UPase–/– mice, of and toxicity in UPase–/– with that in wild type mice. to of UPase–/– to the of the uridine levels and in UPase–/– mice. in these a significantly reduced anesthetic effect the UPase–/– mice. a of in wild type mice, the of of in UPase–/– the of in UPase–/– compared with wild type mice. is an enzyme that the of uridine to uracil. Our and also the of UPase to catalyze the of into with a In UPase–/– mice, the role of UPase in the regulation of uridine homeostasis in and the effect of uridine homeostatic nucleotide metabolism, 5-FU, and These UPase–/– in UPase a that UPase is an gene of the UPase gene in in significant in uridine and pyrimidine nucleotide UPase–/– mice, and UPase activity a abrogation of UPase gene in tissues of uridine activity to the of activity UPase gene and to the presence of in UPase–/– mice. the presence of an to the UPase gene in and as were In is in the kidney, the gene is in the with that a uridine activity of present in the liver of UPase–/– mice, is to In addition, the of uridine in the tissues of UPase–/– the of uridine in the liver and in the gut and UPase–/– mice, the in uridine and pyrimidine nucleotide metabolism were in the of uridine in plasma and tissues and in the of various nucleotide with the loss of the normal pyrimidine and and the a of plasma uridine uridine UPase abrogation also the of as doses of to a 5–6-fold increase in half-life and the of present in wild type mice. in uridine metabolism a of pharmacological doses of In UPase–/– mice, of uridine the plasma uridine concentration to more than more than the plasma uridine concentration in wild type mice. These data indicate an role UPase in uridine metabolism in these UPase–/– that UPase is metabolism in is an of pyrimidine the uridine uridine can to and to pyrimidine and In UPase–/– mice, significant in the concentrations of various pyrimidine total uridine total dCTP, and These changes were present in tissues also although to a lesser extent, an increase in the concentrations of total guanosine total adenosine dGTP, and in the organs. a the changes of the We that to and to the and pyrimidine the of and pyrimidine of the and effect of UPase activity and pyrimidine nucleotide metabolism that UPase a role in the of the uridine and and various and the and in the of is that the of is a in the regulation of these also a to the to increased the of and uridine is an of and pyrimidine normal tissues as and In UPase–/– with plasma and uridine concentrations a significant to 5-FU, the role that uridine in host is that UPase can catalyze the of 5-FU, forming the pyrimidine also to the of the reduced toxicity in UPase–/– mice. the that is to UPase can as a to activity to is a significant in UPase–/– mice, the plasma levels of uridine in these the anesthetic effect of to a and as compared with wild type mice. to the of to the uridine to the the critical role of UPase in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism UPase–/– mice. of pyrimidine metabolism UPase abrogation the and of increased to 5-FU, and reduced to pentobarbital. study the of 5-FU, and and the of the of these UPase is an enzyme that the of uridine to uracil. Our and also the of UPase to catalyze the of into with a In UPase–/– mice, the role of UPase in the regulation of uridine homeostasis in and the effect of uridine homeostatic nucleotide metabolism, 5-FU, and These UPase–/– in UPase a that UPase is an gene of the UPase gene in in significant in uridine and pyrimidine nucleotide In UPase–/– mice, and UPase activity a abrogation of UPase gene in tissues of uridine activity to the of activity UPase gene and to the presence of in UPase–/– mice. the presence of an to the UPase gene in and as were In is in the kidney, the gene is in the with that a uridine activity of present in the liver of UPase–/– mice, is to In addition, the of uridine in the tissues of UPase–/– the of uridine in the liver and in the gut and In UPase–/– mice, the in uridine and pyrimidine nucleotide metabolism were in the of uridine in plasma and tissues and in the of various nucleotide with the loss of the normal pyrimidine and and the a of plasma uridine uridine UPase abrogation also the of as doses of to a 5–6-fold increase in half-life and the of present in wild type mice. in uridine metabolism a of pharmacological doses of In UPase–/– mice, of uridine the plasma uridine concentration to more than more than the plasma uridine concentration in wild type mice. These data indicate an role UPase in uridine metabolism in these UPase–/– that UPase is metabolism in mice. Uridine is an of pyrimidine the uridine uridine can to and to pyrimidine and In UPase–/– mice, significant in the concentrations of various pyrimidine total uridine total dCTP, and These changes were present in tissues also although to a lesser extent, an increase in the concentrations of total guanosine total adenosine dGTP, and in the organs. a the changes of the We that to and to the and pyrimidine the of and pyrimidine of the and effect of UPase activity and pyrimidine nucleotide metabolism that UPase a role in the of the uridine and and various and the and in the of is that the of is a in the regulation of these also a to the to increased the of and uridine is an of and pyrimidine normal tissues as and In UPase–/– with plasma and uridine concentrations a significant to 5-FU, the role that uridine in host is that UPase can catalyze the of 5-FU, forming the pyrimidine also to the of the reduced toxicity in UPase–/– mice. the that is to UPase can as a to activity to Uridine is a significant in UPase–/– mice, the plasma levels of uridine in these the anesthetic effect of to a and as compared with wild type mice. to the of to the uridine to the In the critical role of UPase in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism UPase–/– mice. of pyrimidine metabolism UPase abrogation the and of increased to 5-FU, and reduced to pentobarbital. study the of 5-FU, and and the of the of these