Early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells and requirement of external magnesium for the response.
5-Phosphoribosyl 1-pyrophosphate (PRPP) is a common precursor for the synthesis of all nucleotides and also serves as a critical regulator for the synthesis. In spite of a number of studies in vitro on mammalian PRPP synthetase, our understanding of the regulation of PRPP synthesis in situ is very limited. Various mitogens are known to activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent mouse fibroblasts. We aimed at elucidation of the underlying mechanism for the possible increase in PRPP synthesis in mitogen-stimulated mouse fibroblasts in culture. In order to quantitatively follow metabolic flux through PRPP into nucleotides, [ribosyl-14C]inosine was enzymatically prepared and used as a tracer to preferentially label intracellular ribose phosphate. The radioactivity incorporation into cellular nucleotides was measured. Evidence supported the validity of the method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor (EGF) plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure for PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that EGF plus insulin stimulates the metabolic flux through PRPP. A similar stimulation was induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone. Quiescent Swiss 3T6 cells and human fetal fibroblasts showed a similar stimulation of nucleotide synthesis in response to exposure to serum. For characterization of intracellular signaling pathways, we examined effects of several inhibitors and agents on the stimulation. The divalent cation ionophore A23187 mimicked the response to EGF and insulin in Swiss 3T3 cells, thereby suggesting involvement of divalent cation mobilization in this increase. The effect of the ionophore was not additive to that of the growth factors. Omission of Ca2+ from the incubation medium did not affect the response to EGF and insulin, whereas the omission of Mg2+ did abolish the response. Furosemide, an inhibitor of Mg2+ influx, partially inhibited the stimulated synthesis of nucleotides. Thus, the entry of external Mg2+ into the cells may play a critical role in this signal transduction. These results provided an important access to elucidation of the intracellular mechanisms for the mitogen-induced increase in PRPP and nucleotide syntheses.
Tatibana
M
M
Department of Biochemistry, Chiba University School of Medicine, Japan.
Ishijima
S
S
Kita
K
K
Ishizuka
T
T
Suzuki
N
N
D000818:Animals; D001839:Bombesin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002455:Cell Division / Q000187:drug effects*; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005665:Furosemide / Q000494:pharmacology; D006801:Humans; D007288:Inosine / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008555:Melitten / Q000494:pharmacology; D051379:Mice; D010428:Pentosephosphates / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D012265:Ribonucleotides / Q000096:biosynthesis* / Q000378:metabolism
D002250:Carbon Radioisotopes; D007328:Insulin; D010428:Pentosephosphates; D012265:Ribonucleotides; D008555:Melitten; D000001:Calcimycin; D004533:Egtazic Acid; D007288:Inosine; D004815:Epidermal Growth Factor; D010754:Phosphoribosyl Pyrophosphate; D005665:Furosemide; D001839:Bombesin