Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells.
In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217-228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon chromatographic analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.
Hirai
S
S
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Hayashi
Y
Y
Koizumi
T
T
Nakanishi
N
N
Fukui
T
T
Ichikawa
A
A
D000818:Animals; D001011:Aorta; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism*; D009696:Nucleic Acids / Q000096:biosynthesis; D010430:Pentosyltransferases / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate / Q000096:biosynthesis; D011685:Purine Nucleotides / Q000096:biosynthesis*; D012268:Ribose-Phosphate Pyrophosphokinase / Q000378:metabolism; D013552:Swine
D007042:Hypoxanthines; D009696:Nucleic Acids; D011685:Purine Nucleotides; D019271:Hypoxanthine; D005346:Fibroblast Growth Factors; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D012268:Ribose-Phosphate Pyrophosphokinase