Pharmacological Research, Vol. 25, No . 4, 1992
34
LACK OF INHIBITION OF ORNITHINE DECARBOXYLASE ACTIVITY BY IBUPROFEN J . VESELA, V . SEMECKY and J . DRSATA Departments of Biochemical and Biological Sciences, Faculty of Pharmacy, Charles University, 501 65 Hradec Králové, Czechoslovakia Received in final form 20 November /99/
SUMMARY The influence of the anti-inflammatory drug ibuprofen on the activity of ornithine decarboxylase (ODC, EC 4 .1 .1 .17), the key enzyme of polyamine synthesis, was studied using a 20 000 g supernatant of rat testis and regenerating liver homogenates as sources of the enzyme . Ibuprofen, in all concentrations studied (10 -6 to 2x10-3 M), did not influence either testicular or hepatic ODC activity in vitro . The role of ODC in inflammatory processes and the lack of ODC inhibition by ibuprofen are discussed in view of the controversial findings of other authors . KEY WORDS :
ornithine decarboxylase, ibuprofen, inflammation .
INTRODUCTION Ornithine decarboxylase (ODC, EC 4 .1 .1 .17), by catalysis of decarboxylation of Lornithine, initiates the formation of polyamines de novo and is considered to be the key enzyme in their biosynthesis [1] . Polyamines (putrescine, spermidine, spermine) are necessary to the normal physiological functions of any living cell, they interact with nucleic acids, proteins and biomembranes, and their topical concentration in the cell is sensitively and readily regulated [2] . Regulation can include, according to cell type and the nature of the stimulus, transport of the already formed polyamines in the individual compartments of the cell or transport between the cell and its environment, mutual interconversion of polyamines, and, finally, their biosynthesis de novo, or on the other hand, the pathway of final degradation [3] . An increased need for polyamines, particularly marked in increased proteosynthesis, is often compensated for by their increased biosynthesis de novo, and in such a case an increase in ODC activity is especially marked [4] . When cells are depleted of their polyamines (e .g . using highly specific synthesis inhibitors) they cease to proliferate [5] . Such situations can be induced in certain cells, their groups or whole tissues and organs under various physiological and Correspondence to : Dr J . Drsata .
I043-6618/92/040347-06/$03 .00/0
© 1992 The Italian Pharmacological Society
3 48
Pharmacological Research, Vol . 25, No . 4, 1992
pathological states . ODC activity and polyamine levels are often considered to be the markers of these states . In a previous issue of this journal the results of experiments were published which were to document the influence of S(+) and R(-) enantiomers of ibuprofen on ODC activity in vitro and in vivo [6] . At our laboratory we also investigated ODC and polyamines and examined, among others things, the effect of drugs on ODC activity in vitro and in vivo . One of the drugs under study is ibuprofen and thus in the present paper we communicate our results and our opinions on the possible interpretation of the results of such investigations .
MATERIALS AND METHODS Source of ODC (1) Rat liver regenerating after 2/3 hepatectomy : A rat male of Wistar strain (breeding station, Faculty of Pharmacy), weighing 200 g (water and food ad libitum) was used . Partial hepatectomy was carried out in ether anaesthesia using the method of Higgins and Anderson [7] . The animal was killed 4 .5 h after operation, when the specific activity of ODC is maximal [8] . (2) Decapsulated testis of the adult rat male of Wistar strain of the same breed (weight 350 g, water and food ad libitum) . Processing of the tissue The tissue was homogenized immediately after killing the animal at 0 ° C in an Elvehjem-Potter homogenizer in 0 .1 M sodium phosphate buffer, pH 7 .2 (1 :1, -2 w/v), in which 5x 10-4 M pyridoxal-5'-phosphate, 2x 10 M dithiothreitol and 1 x 10 -'m EDTA .Na2 were dissolved prior to homogenization . The 20000g supernatant was employed to determine enzymatic activity . Determination of ODC activity The modified micromethod of Beaven et al. [9], based on the measurement of the amount of 14 CO 2 released by decarboxylation of [1- 14 C] -L-ornithine, was used . The total volume of incubation mixture was 45 yl, the substrate being [1- 14C]D,L-ornithine (Inst . of Isotopes of the Hungarian Academy of Sciences, Budapest ; specific activity 58 .46 GBq per mol), the final concentrations of D,L-ornithine and pyridoxal-5'-phosphate (the coenzyme) in the incubation mixture were 1x10 -' M and 4 .4x 10-4 M, respectively . After 60 min of incubation at 37 ° C, the reaction was stopped by injecting 0 .1 ml of 2 M citric acid . 14CO2 , released in an enzyme-catalyzed reaction and captured in 30% KOH, was determined by use of an a,ß-spectrometer NE 8302 (Nuclear Enterprises Ltd, Edinburgh), using Bray's scintillation cocktail . Drugs tested Ibuprofen was a gift of the Biomedical Institute, University of Tampere, Finland . DFMO, alpha-difluoromethylornithine, an irreversible inhibitor of ODC, was
Pharmacological Research, Vol. 25, No . 4, 1992
349
obtained by courtesy of the Centre de Recherche Merrel International, Strasbourg, . France . The solution of the drug under study was always prepared extemporaneously by dissolving the drug in 0 .1 M sodium phosphate buffer, pH 7 .2 .
RESULTS The in vitro results of the investigation of the effect of ibuprofen on ODC activity are shown in Table I . Ibuprofen was tested at concentrations in the incubation mixture from 10 -6 to 2x10 -3 M (higher concentrations were not examined because of bad ibuprofen solubility) . Table I includes also the result of the measurement of ODC activity in the presence of DFMO, which is a strong specific irreversible 2 ODC inhibitor . At the concentration used (10_ M) it produces 100% inhibition of the enzyme . This result supplies information on possible decarboxylation of ornithine by other ways than catalysis of ODC, e .g . nonenzymatic decarboxylation or decarboxylation via alternative metabolic routes (see Discussion) . The blank sample illustrates the degree of spontaneous decomposition of [1- 14 C]-ornithine under the given conditions . It is evident that ibuprofen did not decrease in vitro activity of ODC, even at a concentration of 2x10-3 M . The results were the same whether regenerating liver tissue or rat testis tissue were used as the source of the enzyme . The results with DMFO as a highly specific ODC inhibitor show that the activity found in the experiments both with and without ibuprofen actually does represent the result of enzymatic activity of ODC .
Table I In vitro effect of ibuprofen and DFMO on ODC activity of the regenerating liver and testis of the rat Rat tissue
Regenerating liver after partial hepatectomy" Testis"
Concentration of compounds tested
0 2x10-3 M ibuprofen 1 x I O-' M DFMO
ODC activity (in dpm )f' 4CO 2 released)`' --
4712±205 4890±242 84±5
0 550±34 ibuprofen 548±37 i x 10-2 M DFMO 77±6 69±6 Blank`' 0 °20 000 g supernatant of tissue homogenate . 'Phosphate buffer instead of tissue preparation . `Each figure represents the mean±sD of three parallel samples ; details of the ODC assay are in the text. 2x 10-3
M
Pharmacological Research, Vol . 25, No . 4, 1992
350
1 4CO2 putrescine ~ [I - 14 C]- L-ornithine -*L-glutamate gamma-semialdehyde y O O A 1 -pyrroline-5-carboxylase ~ 4C02 I GABA 4- L-gluta mate
I 2-oxoglutarate r4CO succinyl-CoA
Fig. 1 . Scheme of possible metabolic pathways resulting in the release of 14 C0 2 from [1- 14C]L-ornithine . Numbers indicate the following enzymes : (1) ornithine decarboxylase, (2) ornithine-2-oxoacid aminotransferase, (3) 1-pyrroline dehydrogenase, (4) glutamate dehydrogenases (NAD or NADP dependent) or aspartate aminotransferase, (5) 2-oxoglutarate dehydrogenase system, (6) glutamate decarboxylase .
DISCUSSION The results of in vitro experiments in our laboratory performed with the 20 000 g supernatant from the testis of the adult rat male and from the rat liver regenerating after 2/3 hepatectomy have not demonstrated inhibition of ODC, even at an ibuprofen concentration of 2x 10-3 M . These results are different to those of Bruni et al . [6], where ibuprofen is described as an extraordinarily strong inhibitor [in the S(+) enantiomer there is inhibition even at a concentration of the order of 10-8 M, in the R(-) enantiomer at a concentration order of 10-5 M] . There may be several causes of such differences : the two most important in our opinion follow on from the methodology of determination of ODC activity used by Bruni et al . [6] . (1) The generally recommended concentration of the substrate in enzymatic reactions should oscillate within a ten-fold range of the Michaelis constant (Km) . The Km of ODC for L-ornithine ranges between 0 .1 and 0 .2 mm, according to the source of the enzyme [10] . Though Bruni et al . [6] do not mention whether in the in vitro test D,L-ornithine or L-ornithine (the ODC substrate being the L-form only) was used, according to the description of the method of use of L-ornithine the resultant concentration of ODC substrate in the incubation mixture was 1 .2 pm, and in the case of D,L-ornithine was only 0 .6 ,1M . It follows from the characteristics of enzyme kinetics that under these conditions the activity of the enzyme cannot be determined because of an absolute shortage of substrate . The results of the in vitro test therefore obviously cannot document changes in ODC activity due to the drug tested, but they are rather an outcome of analytical inaccuracy . (2) ODC from E . coli was used in [6] in the in vitro tests . Though the ODC molecule is relatively conservative in phylogenetic development, the sequential analysis of the ODC gene from different species reveals sequency homology only in parts of the molecule . The multiplicity of ODC forms from different sources was confirmed by chromatographic, electrophoretic, immunochemical and other studies [4] . The conclusions from the results obtained with the enzyme from a
Pharmacological Research, Vol. 25, No . 4, 1992
35 1
certain source cannot be generalized or applied without verification to another biological species, especially as the species being considered, i .e . E . coli and a mammal, are so far remote from each other . The results of in vitro experiments in [6] are seemingly supported by in vivo experiments in which labelled ornithine was administered to rats . From the viewpoint of the fate of ornithine administered in this way there are several possible metabolic pathways (Fig . 1) . Besides assumed decarboxylation, catalyzed by ODC, 14CO2 can be released from 1- 14C-ornithine after its transformation to glutamic acid by the sequential action of the mitochondrial enzymes ornithine-2oxoacid aminotransferase (EC 2 .6 .1 .13) and 1-pyrroline-5-carboxylate dehydrogenase (EC 1 .5 .1 .12) . Glutamic acid as an intermediate product of ornithine metabolism in the mitochondria can be then converted to 2-oxoglutarate (by either glutamate dehydrogenase, EC 1 .4 .1 .2-1 .4 .1 .4, or aspartate aminotransferase, EC 2 .6 .1 .1) and go through oxidative decarboxylation by a 2oxoglutarate dehydrogenase complex . Glutamic acid can also release carbon dioxide directly, by the action of glutamic acid decarboxylase (GAD, EC 4 .1 .1 .15) with the development of y-aminobutyric acid (GABA) . In addition, the administered ornithine can be included in the intermediary metabolism without releasing 14 CO2 (e .g . in the urea cycle in the liver or via glutamic acid it can be incorporated into proteins, glutathione, etc .) . We presume that decarboxylation catalyzed by ODC will be a minor contributor to all these changes of ornithine . If the in vitro radiometric method, based on the amount of released carbon dioxide from [1- 1Ä C]-ornithine in a time unit, is employed, the possibility of artifacts must always be excluded . The alternative pathways of enzymatic decarboxylation can be excluded or limited by using a pure or partially purified enzyme . Another possibility is to compare the results with those obtained at total inhibition of ODC by DFMO (as carried out in the present paper) . On the other hand, in the in vivo experiments based on the measurement of the amount of 14CO, captured in the metabolic cage, the above-mentioned artifacts can be hardly excluded . Therefore it is impossible to assume that the average activity of ODC in the animal body during the experiment could be determined by this method . In addition, even a perfectly determined average activity of ODC in the whole animal would not provide sufficient information because in different types of cells the basal ODC activity differs markedly [3] . A change in the activity in one type of cell with a higher specific activity can cover a possible opposite change in another type . Besides, ODC responds to most varied stimuli very sensitively and dramatically by changes in its activity (the biological half-life of the enzyme is about 10 min) and in different types of cells it is regulated by different mechanisms . Also, the effect of stress is very marked, e .g . the activity of liver ODC, the basal value of which is relatively high in comparison with other organs [3, 4] increases many times due to its effect . There is another question whether there exists an unambiguous causal relationship between ODC activity and the development of the inflammatory process . It would exist if a change in the enzyme activity were able to produce marked and consistent changes in the concentration of polyamines with the resultant consequences . According to contemporary knowledge it is essentially impossible to achieve depletion of polyamines in the living cell of the organism
352
Pharmacological Research, Vol. 25, No . 4, 1992
merely by ODC inhibition . Compensation reactions are immediately started, polyamines are mobilized from depot bondings, they are transported between the compartments within the cell and the environment, they are mutually interconverted, etc . [3-5] . It is difficult to imagine that in such a complicated process as inflammation depletion of polyamines can be achieved all at once by a mere decrease in ODC activity . Even in the case of clearly demonstrated changes in ODC activity in the individual cell compartments participating in the development of inflammation, these changes will rather be a marker than a key site of possible regulation . The present authors conclude that the possible use of testing changes in ODC activity for the classification of anti-inflammatory drugs seems to be very problematic, and in the case of ibuprofen neither our results nor the results of the Bruni et al . [6] indicate a direct interaction of ODC with this antiphlogistic agent .
ACKNOWLEDGEMENTS The authors wish to thank Dr M . Láznícek from the Radioisotope Laboratory of the Faculty of Pharmacy Hradec Králové for measurement of samples on a liquid scintillation counter.
REFERENCES 1 . Pegg AE, McCann PP . Polyamine metabolism and function in mammalian cells and protozoans . ISI Atlas Sci Biochem 1988 ; 11-18 . 2 . Allen JC . Review : Biochemistry of the polyamines . Cell Biochem Funct 1983 ; 1 : 131-40 . 3 . Seiler N, Heby O . Regulation of cellular polyamines in mammals . Acta Biochim Biophys Hung 1988 ; 23 : 1-36. 4 . Heby O, Persson L . Molecular genetics of polyamine synthesis in eukaryotic cells . TIBS 1990;15 :153-8 . 5 . Porter CW, Sufrin JR . Interference with polyamine biosynthesis and/or function by analogs of polyamines or methionine as a potential anti-cancer chemotherapeutic strategy . Anticancer Res 1986 ; 6 : 525-42 . 6 . Bruni G, Runci FM, Fiaschi AI, Segre G . Inhibition of ornithine-decarboxylase produced by S(+) and R(-) ibuprofen in rats . Pharmacol Res 1990 ; 22 : 97-102 . 7 . Higgins GM, Anderson RM . Experimental pathology of the liver . 1 . Restoration of the liver of the white rat following partial surgical removal . Arch Pathol 1931 ; 12 : 186-202 . 8 . Veselá J, Drsata J . Effect of 5-[2-(N,N-dimethyl-amino)-ethoxy]-7-oxo-7Hbenzo(c)fluorene hydrochloride (the cytostatic agent Benfluron) on the activity of ornithine decarboxylase of the regenerating rat liver in vivo . Cesk Farm 1988 : 37 : 251-4 . 9 . Beaven MA, Wilcox G, Terpstra GK . A microprocedure for the measurement of 14CO, release from [ 14C]-carboxyl-labeled amino acids . Anal Biochem 1978 ; 84 : 638--41 . 10 . Obenrader MF, Prouty WF . Detection of multiple forms of rat liver ornithine decarboxylase . J Biol Chem 1977 ; 252 : 2860-5 .
34
LACK OF INHIBITION OF ORNITHINE DECARBOXYLASE ACTIVITY BY IBUPROFEN J . VESELA, V . SEMECKY and J . DRSATA Departments of Biochemical and Biological Sciences, Faculty of Pharmacy, Charles University, 501 65 Hradec Králové, Czechoslovakia Received in final form 20 November /99/
SUMMARY The influence of the anti-inflammatory drug ibuprofen on the activity of ornithine decarboxylase (ODC, EC 4 .1 .1 .17), the key enzyme of polyamine synthesis, was studied using a 20 000 g supernatant of rat testis and regenerating liver homogenates as sources of the enzyme . Ibuprofen, in all concentrations studied (10 -6 to 2x10-3 M), did not influence either testicular or hepatic ODC activity in vitro . The role of ODC in inflammatory processes and the lack of ODC inhibition by ibuprofen are discussed in view of the controversial findings of other authors . KEY WORDS :
ornithine decarboxylase, ibuprofen, inflammation .
INTRODUCTION Ornithine decarboxylase (ODC, EC 4 .1 .1 .17), by catalysis of decarboxylation of Lornithine, initiates the formation of polyamines de novo and is considered to be the key enzyme in their biosynthesis [1] . Polyamines (putrescine, spermidine, spermine) are necessary to the normal physiological functions of any living cell, they interact with nucleic acids, proteins and biomembranes, and their topical concentration in the cell is sensitively and readily regulated [2] . Regulation can include, according to cell type and the nature of the stimulus, transport of the already formed polyamines in the individual compartments of the cell or transport between the cell and its environment, mutual interconversion of polyamines, and, finally, their biosynthesis de novo, or on the other hand, the pathway of final degradation [3] . An increased need for polyamines, particularly marked in increased proteosynthesis, is often compensated for by their increased biosynthesis de novo, and in such a case an increase in ODC activity is especially marked [4] . When cells are depleted of their polyamines (e .g . using highly specific synthesis inhibitors) they cease to proliferate [5] . Such situations can be induced in certain cells, their groups or whole tissues and organs under various physiological and Correspondence to : Dr J . Drsata .
I043-6618/92/040347-06/$03 .00/0
© 1992 The Italian Pharmacological Society
3 48
Pharmacological Research, Vol . 25, No . 4, 1992
pathological states . ODC activity and polyamine levels are often considered to be the markers of these states . In a previous issue of this journal the results of experiments were published which were to document the influence of S(+) and R(-) enantiomers of ibuprofen on ODC activity in vitro and in vivo [6] . At our laboratory we also investigated ODC and polyamines and examined, among others things, the effect of drugs on ODC activity in vitro and in vivo . One of the drugs under study is ibuprofen and thus in the present paper we communicate our results and our opinions on the possible interpretation of the results of such investigations .
MATERIALS AND METHODS Source of ODC (1) Rat liver regenerating after 2/3 hepatectomy : A rat male of Wistar strain (breeding station, Faculty of Pharmacy), weighing 200 g (water and food ad libitum) was used . Partial hepatectomy was carried out in ether anaesthesia using the method of Higgins and Anderson [7] . The animal was killed 4 .5 h after operation, when the specific activity of ODC is maximal [8] . (2) Decapsulated testis of the adult rat male of Wistar strain of the same breed (weight 350 g, water and food ad libitum) . Processing of the tissue The tissue was homogenized immediately after killing the animal at 0 ° C in an Elvehjem-Potter homogenizer in 0 .1 M sodium phosphate buffer, pH 7 .2 (1 :1, -2 w/v), in which 5x 10-4 M pyridoxal-5'-phosphate, 2x 10 M dithiothreitol and 1 x 10 -'m EDTA .Na2 were dissolved prior to homogenization . The 20000g supernatant was employed to determine enzymatic activity . Determination of ODC activity The modified micromethod of Beaven et al. [9], based on the measurement of the amount of 14 CO 2 released by decarboxylation of [1- 14 C] -L-ornithine, was used . The total volume of incubation mixture was 45 yl, the substrate being [1- 14C]D,L-ornithine (Inst . of Isotopes of the Hungarian Academy of Sciences, Budapest ; specific activity 58 .46 GBq per mol), the final concentrations of D,L-ornithine and pyridoxal-5'-phosphate (the coenzyme) in the incubation mixture were 1x10 -' M and 4 .4x 10-4 M, respectively . After 60 min of incubation at 37 ° C, the reaction was stopped by injecting 0 .1 ml of 2 M citric acid . 14CO2 , released in an enzyme-catalyzed reaction and captured in 30% KOH, was determined by use of an a,ß-spectrometer NE 8302 (Nuclear Enterprises Ltd, Edinburgh), using Bray's scintillation cocktail . Drugs tested Ibuprofen was a gift of the Biomedical Institute, University of Tampere, Finland . DFMO, alpha-difluoromethylornithine, an irreversible inhibitor of ODC, was
Pharmacological Research, Vol. 25, No . 4, 1992
349
obtained by courtesy of the Centre de Recherche Merrel International, Strasbourg, . France . The solution of the drug under study was always prepared extemporaneously by dissolving the drug in 0 .1 M sodium phosphate buffer, pH 7 .2 .
RESULTS The in vitro results of the investigation of the effect of ibuprofen on ODC activity are shown in Table I . Ibuprofen was tested at concentrations in the incubation mixture from 10 -6 to 2x10 -3 M (higher concentrations were not examined because of bad ibuprofen solubility) . Table I includes also the result of the measurement of ODC activity in the presence of DFMO, which is a strong specific irreversible 2 ODC inhibitor . At the concentration used (10_ M) it produces 100% inhibition of the enzyme . This result supplies information on possible decarboxylation of ornithine by other ways than catalysis of ODC, e .g . nonenzymatic decarboxylation or decarboxylation via alternative metabolic routes (see Discussion) . The blank sample illustrates the degree of spontaneous decomposition of [1- 14 C]-ornithine under the given conditions . It is evident that ibuprofen did not decrease in vitro activity of ODC, even at a concentration of 2x10-3 M . The results were the same whether regenerating liver tissue or rat testis tissue were used as the source of the enzyme . The results with DMFO as a highly specific ODC inhibitor show that the activity found in the experiments both with and without ibuprofen actually does represent the result of enzymatic activity of ODC .
Table I In vitro effect of ibuprofen and DFMO on ODC activity of the regenerating liver and testis of the rat Rat tissue
Regenerating liver after partial hepatectomy" Testis"
Concentration of compounds tested
0 2x10-3 M ibuprofen 1 x I O-' M DFMO
ODC activity (in dpm )f' 4CO 2 released)`' --
4712±205 4890±242 84±5
0 550±34 ibuprofen 548±37 i x 10-2 M DFMO 77±6 69±6 Blank`' 0 °20 000 g supernatant of tissue homogenate . 'Phosphate buffer instead of tissue preparation . `Each figure represents the mean±sD of three parallel samples ; details of the ODC assay are in the text. 2x 10-3
M
Pharmacological Research, Vol . 25, No . 4, 1992
350
1 4CO2 putrescine ~ [I - 14 C]- L-ornithine -*L-glutamate gamma-semialdehyde y O O A 1 -pyrroline-5-carboxylase ~ 4C02 I GABA 4- L-gluta mate
I 2-oxoglutarate r4CO succinyl-CoA
Fig. 1 . Scheme of possible metabolic pathways resulting in the release of 14 C0 2 from [1- 14C]L-ornithine . Numbers indicate the following enzymes : (1) ornithine decarboxylase, (2) ornithine-2-oxoacid aminotransferase, (3) 1-pyrroline dehydrogenase, (4) glutamate dehydrogenases (NAD or NADP dependent) or aspartate aminotransferase, (5) 2-oxoglutarate dehydrogenase system, (6) glutamate decarboxylase .
DISCUSSION The results of in vitro experiments in our laboratory performed with the 20 000 g supernatant from the testis of the adult rat male and from the rat liver regenerating after 2/3 hepatectomy have not demonstrated inhibition of ODC, even at an ibuprofen concentration of 2x 10-3 M . These results are different to those of Bruni et al . [6], where ibuprofen is described as an extraordinarily strong inhibitor [in the S(+) enantiomer there is inhibition even at a concentration of the order of 10-8 M, in the R(-) enantiomer at a concentration order of 10-5 M] . There may be several causes of such differences : the two most important in our opinion follow on from the methodology of determination of ODC activity used by Bruni et al . [6] . (1) The generally recommended concentration of the substrate in enzymatic reactions should oscillate within a ten-fold range of the Michaelis constant (Km) . The Km of ODC for L-ornithine ranges between 0 .1 and 0 .2 mm, according to the source of the enzyme [10] . Though Bruni et al . [6] do not mention whether in the in vitro test D,L-ornithine or L-ornithine (the ODC substrate being the L-form only) was used, according to the description of the method of use of L-ornithine the resultant concentration of ODC substrate in the incubation mixture was 1 .2 pm, and in the case of D,L-ornithine was only 0 .6 ,1M . It follows from the characteristics of enzyme kinetics that under these conditions the activity of the enzyme cannot be determined because of an absolute shortage of substrate . The results of the in vitro test therefore obviously cannot document changes in ODC activity due to the drug tested, but they are rather an outcome of analytical inaccuracy . (2) ODC from E . coli was used in [6] in the in vitro tests . Though the ODC molecule is relatively conservative in phylogenetic development, the sequential analysis of the ODC gene from different species reveals sequency homology only in parts of the molecule . The multiplicity of ODC forms from different sources was confirmed by chromatographic, electrophoretic, immunochemical and other studies [4] . The conclusions from the results obtained with the enzyme from a
Pharmacological Research, Vol. 25, No . 4, 1992
35 1
certain source cannot be generalized or applied without verification to another biological species, especially as the species being considered, i .e . E . coli and a mammal, are so far remote from each other . The results of in vitro experiments in [6] are seemingly supported by in vivo experiments in which labelled ornithine was administered to rats . From the viewpoint of the fate of ornithine administered in this way there are several possible metabolic pathways (Fig . 1) . Besides assumed decarboxylation, catalyzed by ODC, 14CO2 can be released from 1- 14C-ornithine after its transformation to glutamic acid by the sequential action of the mitochondrial enzymes ornithine-2oxoacid aminotransferase (EC 2 .6 .1 .13) and 1-pyrroline-5-carboxylate dehydrogenase (EC 1 .5 .1 .12) . Glutamic acid as an intermediate product of ornithine metabolism in the mitochondria can be then converted to 2-oxoglutarate (by either glutamate dehydrogenase, EC 1 .4 .1 .2-1 .4 .1 .4, or aspartate aminotransferase, EC 2 .6 .1 .1) and go through oxidative decarboxylation by a 2oxoglutarate dehydrogenase complex . Glutamic acid can also release carbon dioxide directly, by the action of glutamic acid decarboxylase (GAD, EC 4 .1 .1 .15) with the development of y-aminobutyric acid (GABA) . In addition, the administered ornithine can be included in the intermediary metabolism without releasing 14 CO2 (e .g . in the urea cycle in the liver or via glutamic acid it can be incorporated into proteins, glutathione, etc .) . We presume that decarboxylation catalyzed by ODC will be a minor contributor to all these changes of ornithine . If the in vitro radiometric method, based on the amount of released carbon dioxide from [1- 1Ä C]-ornithine in a time unit, is employed, the possibility of artifacts must always be excluded . The alternative pathways of enzymatic decarboxylation can be excluded or limited by using a pure or partially purified enzyme . Another possibility is to compare the results with those obtained at total inhibition of ODC by DFMO (as carried out in the present paper) . On the other hand, in the in vivo experiments based on the measurement of the amount of 14CO, captured in the metabolic cage, the above-mentioned artifacts can be hardly excluded . Therefore it is impossible to assume that the average activity of ODC in the animal body during the experiment could be determined by this method . In addition, even a perfectly determined average activity of ODC in the whole animal would not provide sufficient information because in different types of cells the basal ODC activity differs markedly [3] . A change in the activity in one type of cell with a higher specific activity can cover a possible opposite change in another type . Besides, ODC responds to most varied stimuli very sensitively and dramatically by changes in its activity (the biological half-life of the enzyme is about 10 min) and in different types of cells it is regulated by different mechanisms . Also, the effect of stress is very marked, e .g . the activity of liver ODC, the basal value of which is relatively high in comparison with other organs [3, 4] increases many times due to its effect . There is another question whether there exists an unambiguous causal relationship between ODC activity and the development of the inflammatory process . It would exist if a change in the enzyme activity were able to produce marked and consistent changes in the concentration of polyamines with the resultant consequences . According to contemporary knowledge it is essentially impossible to achieve depletion of polyamines in the living cell of the organism
352
Pharmacological Research, Vol. 25, No . 4, 1992
merely by ODC inhibition . Compensation reactions are immediately started, polyamines are mobilized from depot bondings, they are transported between the compartments within the cell and the environment, they are mutually interconverted, etc . [3-5] . It is difficult to imagine that in such a complicated process as inflammation depletion of polyamines can be achieved all at once by a mere decrease in ODC activity . Even in the case of clearly demonstrated changes in ODC activity in the individual cell compartments participating in the development of inflammation, these changes will rather be a marker than a key site of possible regulation . The present authors conclude that the possible use of testing changes in ODC activity for the classification of anti-inflammatory drugs seems to be very problematic, and in the case of ibuprofen neither our results nor the results of the Bruni et al . [6] indicate a direct interaction of ODC with this antiphlogistic agent .
ACKNOWLEDGEMENTS The authors wish to thank Dr M . Láznícek from the Radioisotope Laboratory of the Faculty of Pharmacy Hradec Králové for measurement of samples on a liquid scintillation counter.
REFERENCES 1 . Pegg AE, McCann PP . Polyamine metabolism and function in mammalian cells and protozoans . ISI Atlas Sci Biochem 1988 ; 11-18 . 2 . Allen JC . Review : Biochemistry of the polyamines . Cell Biochem Funct 1983 ; 1 : 131-40 . 3 . Seiler N, Heby O . Regulation of cellular polyamines in mammals . Acta Biochim Biophys Hung 1988 ; 23 : 1-36. 4 . Heby O, Persson L . Molecular genetics of polyamine synthesis in eukaryotic cells . TIBS 1990;15 :153-8 . 5 . Porter CW, Sufrin JR . Interference with polyamine biosynthesis and/or function by analogs of polyamines or methionine as a potential anti-cancer chemotherapeutic strategy . Anticancer Res 1986 ; 6 : 525-42 . 6 . Bruni G, Runci FM, Fiaschi AI, Segre G . Inhibition of ornithine-decarboxylase produced by S(+) and R(-) ibuprofen in rats . Pharmacol Res 1990 ; 22 : 97-102 . 7 . Higgins GM, Anderson RM . Experimental pathology of the liver . 1 . Restoration of the liver of the white rat following partial surgical removal . Arch Pathol 1931 ; 12 : 186-202 . 8 . Veselá J, Drsata J . Effect of 5-[2-(N,N-dimethyl-amino)-ethoxy]-7-oxo-7Hbenzo(c)fluorene hydrochloride (the cytostatic agent Benfluron) on the activity of ornithine decarboxylase of the regenerating rat liver in vivo . Cesk Farm 1988 : 37 : 251-4 . 9 . Beaven MA, Wilcox G, Terpstra GK . A microprocedure for the measurement of 14CO, release from [ 14C]-carboxyl-labeled amino acids . Anal Biochem 1978 ; 84 : 638--41 . 10 . Obenrader MF, Prouty WF . Detection of multiple forms of rat liver ornithine decarboxylase . J Biol Chem 1977 ; 252 : 2860-5 .
