Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid.
Tetrapyrrole synthesis in CNCM-1221 cells exposed to 0.6 mM aminolaevulinic acid (ALA) was found to be approximately linear over a 6-h period of incubation. The rate was not significantly affected by cell density over a range of 0.015 to 0.15 x 10(6) cells cm(-2) (final cell density). Tetrapyrrole synthesis was not affected by GABA or glutamic acid in concentrations up to 6 mM and 2.72 mM respectively, suggesting that these amino acids, which are similar in structure to ALA, do not competitively inhibit the ALA uptake pathway in these cells. Pre-exposure to haem arginate (up to 100 microM) was inhibitory, presumably by suppression (through the inhibition of ALA synthase) of an endogenous component of the response. The ALA-stimulated response was not modified by co-exposure to AIA (up to 100 mg ml(-1)). Despite significant reduction of protein synthesis, the porphyrinogenic response of cells exposed to ALA was unaffected by cycloheximide (10 microg ml(-1)) or actinomycin D (10 microg ml(-1)) even when cells were preincubated with these agents for 3 h before ALA exposure. Fetal bovine serum (10%) inhibited tetrapyrrole synthesis by 30% but increased the rate of porphyrin export by cells by a factor of 1.5. The uptake of [14C]ALA was shown to be strongly influenced by the density of the cultures. In dense cultures (final cell density of approximately 0.15 x 10(6) cells cm(-2)), the ALA uptake rate was less than 0.8 compared with a maximum rate of 4.2 fmol per cell h(-1) at a cell density of 0.02 x 10(6) cells cm(-2). Since tetrapyrrole synthesis is less affected than ALA uptake by cell density, the resultant discrepancy in ALA incorporation occurring in dense cultures implies that endogenous ALA synthesis is induced in these cells. ALA uptake was not affected by cycloheximide or actinomycin D in serum-free conditions. However, fetal bovine serum decreased external ALA uptake by about 50%. This effect was abrogated by preincubation with cycloheximide.
Washbrook
R
R
Department of Molecular Pathology, UCL Medical School, London, UK.
Fukuda
H
H
Battle
A
A
Riley
P
P
D000622:Aminolevulinic Acid / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000494:pharmacology; D001692:Biological Transport; D002250:Carbon Radioisotopes; D002417:Cattle; D002455:Cell Division; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology; D006418:Heme / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008322:Mammals; D011758:Pyrroles / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D045725:Tetrapyrroles; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002250:Carbon Radioisotopes; D011758:Pyrroles; D045725:Tetrapyrroles; D003609:Dactinomycin; D018698:Glutamic Acid; D006418:Heme; D005680:gamma-Aminobutyric Acid; D000622:Aminolevulinic Acid; D001120:Arginine; D003513:Cycloheximide; D007930:Leucine; C048849:heme arginate