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Allyglycine affects acetylation of putrescine and spermidine in mouse brain
Neuropharmacology Vol. 23, No. 3, pp. 387-390, 1984 Printed in Great Britain
0028-3908/84 $3.00 + 0.00 Pergamon Press Ltd
PRELIMINARY Reprinted
NOTES
from Vol. 22, No. 10, pp. 1237-1239
ALLYGLYCI NE AFFECTS ACETYLATION
OF PUTRESCINE AND SPERMIDI NE
IN MOUSE BRAIN J. G. Ortiz*, Laboratory
E. Giacobini**,
T. Schmidt-Glenewinkel***
of Neuropsychopharmacology, Department of Biobehavioral University of Connecticut, Storrs, CT 06268, USA
Science,
(Accepted 1 Augu.& 1983) SUMMARY - Administration of allylglycine to mice ( .8 mmole/kg, i. p. ) results in a depletion of GABA levels, and it is accompanied by a decrease in SAM-DC activity and spermidine and spermine levels (Pajunen et al., 1979). Here we describe a biphasic effect on the acetylation of putrescine and spermidine in mouse brain homogenate. There appears to be an inverse correlation between the initial decrease in spermidine levels at 2 hours and the increase in the acetylation of spermidine. This ‘s suggestive of a conversion of spermidine, probably through N‘i -acetylspermidine to putrescine. The peak of putrescine acetylation observed by us at 4 hours may also reflect a conversion of putrescine, via acetylputrescine to GABA. The interconversion hypothesis is supported by the fact that putrescine levels remain essentially stable in spite of a significant depletion of spermidine and spermine. In addition, there is a decrease in putrescine and spermidine acetylation at 8 hours, which coincides with the increase in ODC activity and the increase towards control levels of GAD activity (Pajunen et al,, 1979). Such inverse correlations suggest a mechanism for replenishment of polyamines once GAD activity returns to control levels. In the brain, acetylation of putrescine may be rate-limiting in its conversion to GABA (Seiler, Schmidt-Glenewinkel and Sarhan, 1979; Seiler, 1980). This pathway is particularly active during neural development (DeMello et al., 1976; Sobue and Nakajima, 1978), however as the animal matures, the contribution of putrescine to GABA levels decreases. We found the acetylation of polyamines to be high during development (Ortiz, Giacobini and Schmidt-Glenewinkel, 1983). Furthermore, we found polyamine acetylation in the P and cytosolic fractions of the developing mouse brain, but not in the adult (Or 0.12, Giacobini and SchmidtGlenewinkel, 198313). In the present paper, we examine the acetylation of putrescine after the administration of allylglycine, a GAD (glutamic acid decarboxylase) inhibitor, in order to assess whether the putrescine to GABA pathway could act as a compensatory mechanism. We also examine the acetylation of spermidine, since putrescine levels are 10~ in the adult brain (Seiler and Schmidt-Glenewidel, could contribute to the putrescine 1975) and spermidine, via N1-acetylspermidine, levels (Bolkenius and Seiler, 1981).
Key Words: putrescine, spermidine, acetylation, mouse brain, GABA, allylglycine. *Present address: Department of Pharmacology, Medical Sciences Campus, G. P. 0. Box 5067, San Juan, Puerto Rico 00936. **Present address: Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL 62708, USA. ***Present address: Albert Einstein College of Medicine, Department of Developmental Biology and Cancer, Bronx, NY 10461, USA. 387
Preliminary
Notes
METHODS Chemicals. Putrescine. 2HC1, spermidine. 3HC1, pargyline. HCl, hydroxylamine. HCl, acetylCoA (Li-salt), 2-mercaptoethanol, and ally lycine were obtained from Sigma Chemical Co. (St. Louis, MO). l-[ 1s C] acetylCoA, 50-65 mCi/mmole, was from ICN pharmaceuticals (Irvine, CA). C57B1/6Bg mice, 2.5-3 month old (25-30 g) were from the SPF colony of this department. Allyglycine was administered i. p. (. 8 mmole/kg). Animals were sacrificed by decapitation and their brains frozen (overnight) at -90” C. The tissue was homogenized in 100 mM sodium phosphate buffer, pH, 8.0 (1:6, w/v). Enzymatic assay. 50 plof homogenate were preincubated with 0.3 mM pargyline and 1.6 mM 2-mercaptoethanol for 5 min at 37” C. Then, 5 . mM polyamine and 50 PM acetylCoA (26,000 dpm/sample) were added to a final volume of 100 ~1 and incubated for another 10 min. The reaction was terminated by the addition of 20 kmoles of hydroxylamine in 0.1 N HCl and boiling for 5 min. Boiled tissue blanks were used in all the experiments. The reaction product was quantified using cellulose phosphate discs (2.3 cm, Whatman P-81) as described by Libby (1978). Further characterization of the assay has been presented elsewhere (Ortiz et al., 1983a). RESULTS AND DISCUSSION The effect of allyglycine administration on the acetylation of putrescine and spermidine in mouse brain homogenates is shown in Fig. 1. Relative to 0 hour levels, acetylation of spermidine peaks at 2 hours, and returns to control levels by 6 hours. Spermidine acetylase activity decreases at 8 hours and returns to control levels by 10 hours. A similar biphasic curve is observed for the acetylation of putrescine, except that putrescine acetylation peaks at 4 hours. Saline injected animals were compared at 10 hours. In these animals, putrescine and spermidine acetylase activity was not different from 0 time. Furthermore, we were unable to detect any changes in polyamine acetylase activities in brain homogenates after i. p. administration of saline. The initial rise in putrescine and spermidine acetylation observed by us after the administration of allylglycine correlates well with the decrease in GAD, and SAM-DC (S-adenosylmethionine decarboxylase) activities, and GABA, spermidine and spermine levels observed by Pajunen et al. (1979). It is possible that the different peaks of putresc’ne and spermidine acetylation correspond to separate enzymes. Spermidine N5 -acetyltransferase appears to be rate-limiting in the conversion of spermidine to putrescine (Bolkenius and Seiler, 1981), however, it does not act on putrescine (Matsui and Pegg, 1980). There is an inverse correlation between the decrease in spermidine and putrescine acetylation which we observe at 8 hours and the peak of ODC (ornithine decarboxylase) activity reported by Pajunen et al. (1979). This inverse correlation suggests a mechanism by which polyamine levels are replenished. Such a hypothesis is supported by a concomitant increase in SAM-DC and GAD activities. Pajunen et al. (1979), were unable to detect differences in the formation of GABA from putrescine in control vs. allyglycine-treated mice. The use of 10 ~1 intraventricular injections in the mouse with distilled water as a vehicle as well as the large variations in specific activity, made such results hardly conclusive. Furthermore, this study (Pajunen et al., 1979) did not account for changes in polyamine metabolism, nor investigated formation of other possible polyamine metabolites, such as Y -glutamylputrescine or homocarnosine (Konishi et al., 1977).
Preliminary
TIME
Notes
AFTER
389
INJECTION
+...
PUTRESCINE
-+-
SPERMIDINE
(hr)
The actual formation of GABA from putrescine after inhibition of GAD, as well as the presence of acetylspermidine isomer(s) formed under such conditions, Furthermore, it is not known whether GABA derived remain to be investigated. from putrescine participates in neurotransmission. In conclusion, depletion of GABA levels, brought about by the administration of allylglycine, results in a biphasic change in the acetylation of putrescine and The initial increase in putrescine and spermidine acetylation spcrmidine. correlates well with a decrease in spermidine, spermine and GABA levels, on one hand, and with GAD and SAM-DC activities, on the other. The decrease in acetylation of putrescine and spermidine observed by us at 8 hours coincides with the peak of ODC activity and the return towards control values of GAD and SAMDC activities and of GABA and spermidine levels.
390
Preliminary
Notes
These results, as well as the presence of polyamine acetylation (Ortiz, Giacobini and Schmidt-Glenewinkel, 1983b) in the P2 and cytosolic fractions of the developing mouse brain, support the hypothesis of a contribution of putrescine to GABA levels during development, and possibly during periods of GABA depletion. ACKNOWLEDGEMENT J. G. 0. was a recipient of a predoctoral fellowship from the University of This work was supported by grants NS 11430 and NS 14086 to E. G. Connecticut.
REFERENCES BOLKENIUS, F. N. and SEILER, N. (1981) Acetyl derivatives in polyamine catabolism, Intl. J. Biochem. 13:287-292.
as intermediates
DEMELLO, F.G., BACHRACH, U. and NIREMBERG, M. (1976) Ornithine and glutamic acid decarboxylase activities in the developing chick retina, J. Neurochem. 27:847-851. KONISHI, H., NAKAJIMA, T. and SANO, I. (1977) Metabolism the central nervous system, J. Biochem. 81:355-360.
of putrescine
in
LIBBY, P.R. (1978) Calf-liver nuclear N-acetyltransferases: (purification and properties of two enzymes with both spermidine acetyltransferase and histone acetyltransferase activities), J. Biol. Chem. 235:233-237. MATSUI, I. and PEGG, A. E. (1980) Increase in the acetylation of spermidine in rat liver extracts brought about by treatment with carbon tetrachloride, Biochem. Biophys. Res. Commun. pp. 1009-1015. ORTIZ, J. G., GIACOBINI, E. and SCHMIDT-GLENEWINKEL, T. (1983a) Acetylation of polyamines in mouse brain: Subcellular and regional distribution, J. Neuroscience Res. 9:193-201. ORTIZ, J.G., GIACOBINI, E. and SCHMIDT-GLENEWINKEL, T. (198313) Polyamine acetylation in the developing and aging mouse brain, Intl. J. Developmental Neuroscience, 1: 179-185. PAJUNEN, A. E. I., HIETALA, O.A., BARUCH-VIRRANSALO, E. L. and PIHA, S. S. (1979) The effect of DL-allylglycine on polyamine and GABA metabolism in mouse brain, J. Neurochem. 32:1401-1408. SEILER, N. (1980) On the role of GABA in vertebrate Physiol. Chem. and Phys. 12:411-429.
polyamine metabolism,
SEILER, N., SCHMIDT-GLENEWINKEL, T. and SARHAN, S. (1979) On the formation of y -aminobutyric acid from putrescine in brain, J. Biochem. 86:277-278. SEILER, N. and SCHMIDT-GLENEWINKEL, T. (1975) Regional distribution of putrescine, spermidine and spermine in relation to the distribution of RNA and DNA in the rat nervous system, J. Neurochem. 24:791-795. SOBUE, K. and NAKAJIMA, T. (1978) Change in the concentrations of polyamines and Y -aminobutyric acid and their formation in chick embryo brain during development, J. Neurochem. 30:2’77-279.
0028-3908/84 $3.00 + 0.00 Pergamon Press Ltd
PRELIMINARY Reprinted
NOTES
from Vol. 22, No. 10, pp. 1237-1239
ALLYGLYCI NE AFFECTS ACETYLATION
OF PUTRESCINE AND SPERMIDI NE
IN MOUSE BRAIN J. G. Ortiz*, Laboratory
E. Giacobini**,
T. Schmidt-Glenewinkel***
of Neuropsychopharmacology, Department of Biobehavioral University of Connecticut, Storrs, CT 06268, USA
Science,
(Accepted 1 Augu.& 1983) SUMMARY - Administration of allylglycine to mice ( .8 mmole/kg, i. p. ) results in a depletion of GABA levels, and it is accompanied by a decrease in SAM-DC activity and spermidine and spermine levels (Pajunen et al., 1979). Here we describe a biphasic effect on the acetylation of putrescine and spermidine in mouse brain homogenate. There appears to be an inverse correlation between the initial decrease in spermidine levels at 2 hours and the increase in the acetylation of spermidine. This ‘s suggestive of a conversion of spermidine, probably through N‘i -acetylspermidine to putrescine. The peak of putrescine acetylation observed by us at 4 hours may also reflect a conversion of putrescine, via acetylputrescine to GABA. The interconversion hypothesis is supported by the fact that putrescine levels remain essentially stable in spite of a significant depletion of spermidine and spermine. In addition, there is a decrease in putrescine and spermidine acetylation at 8 hours, which coincides with the increase in ODC activity and the increase towards control levels of GAD activity (Pajunen et al,, 1979). Such inverse correlations suggest a mechanism for replenishment of polyamines once GAD activity returns to control levels. In the brain, acetylation of putrescine may be rate-limiting in its conversion to GABA (Seiler, Schmidt-Glenewinkel and Sarhan, 1979; Seiler, 1980). This pathway is particularly active during neural development (DeMello et al., 1976; Sobue and Nakajima, 1978), however as the animal matures, the contribution of putrescine to GABA levels decreases. We found the acetylation of polyamines to be high during development (Ortiz, Giacobini and Schmidt-Glenewinkel, 1983). Furthermore, we found polyamine acetylation in the P and cytosolic fractions of the developing mouse brain, but not in the adult (Or 0.12, Giacobini and SchmidtGlenewinkel, 198313). In the present paper, we examine the acetylation of putrescine after the administration of allylglycine, a GAD (glutamic acid decarboxylase) inhibitor, in order to assess whether the putrescine to GABA pathway could act as a compensatory mechanism. We also examine the acetylation of spermidine, since putrescine levels are 10~ in the adult brain (Seiler and Schmidt-Glenewidel, could contribute to the putrescine 1975) and spermidine, via N1-acetylspermidine, levels (Bolkenius and Seiler, 1981).
Key Words: putrescine, spermidine, acetylation, mouse brain, GABA, allylglycine. *Present address: Department of Pharmacology, Medical Sciences Campus, G. P. 0. Box 5067, San Juan, Puerto Rico 00936. **Present address: Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL 62708, USA. ***Present address: Albert Einstein College of Medicine, Department of Developmental Biology and Cancer, Bronx, NY 10461, USA. 387
Preliminary
Notes
METHODS Chemicals. Putrescine. 2HC1, spermidine. 3HC1, pargyline. HCl, hydroxylamine. HCl, acetylCoA (Li-salt), 2-mercaptoethanol, and ally lycine were obtained from Sigma Chemical Co. (St. Louis, MO). l-[ 1s C] acetylCoA, 50-65 mCi/mmole, was from ICN pharmaceuticals (Irvine, CA). C57B1/6Bg mice, 2.5-3 month old (25-30 g) were from the SPF colony of this department. Allyglycine was administered i. p. (. 8 mmole/kg). Animals were sacrificed by decapitation and their brains frozen (overnight) at -90” C. The tissue was homogenized in 100 mM sodium phosphate buffer, pH, 8.0 (1:6, w/v). Enzymatic assay. 50 plof homogenate were preincubated with 0.3 mM pargyline and 1.6 mM 2-mercaptoethanol for 5 min at 37” C. Then, 5 . mM polyamine and 50 PM acetylCoA (26,000 dpm/sample) were added to a final volume of 100 ~1 and incubated for another 10 min. The reaction was terminated by the addition of 20 kmoles of hydroxylamine in 0.1 N HCl and boiling for 5 min. Boiled tissue blanks were used in all the experiments. The reaction product was quantified using cellulose phosphate discs (2.3 cm, Whatman P-81) as described by Libby (1978). Further characterization of the assay has been presented elsewhere (Ortiz et al., 1983a). RESULTS AND DISCUSSION The effect of allyglycine administration on the acetylation of putrescine and spermidine in mouse brain homogenates is shown in Fig. 1. Relative to 0 hour levels, acetylation of spermidine peaks at 2 hours, and returns to control levels by 6 hours. Spermidine acetylase activity decreases at 8 hours and returns to control levels by 10 hours. A similar biphasic curve is observed for the acetylation of putrescine, except that putrescine acetylation peaks at 4 hours. Saline injected animals were compared at 10 hours. In these animals, putrescine and spermidine acetylase activity was not different from 0 time. Furthermore, we were unable to detect any changes in polyamine acetylase activities in brain homogenates after i. p. administration of saline. The initial rise in putrescine and spermidine acetylation observed by us after the administration of allylglycine correlates well with the decrease in GAD, and SAM-DC (S-adenosylmethionine decarboxylase) activities, and GABA, spermidine and spermine levels observed by Pajunen et al. (1979). It is possible that the different peaks of putresc’ne and spermidine acetylation correspond to separate enzymes. Spermidine N5 -acetyltransferase appears to be rate-limiting in the conversion of spermidine to putrescine (Bolkenius and Seiler, 1981), however, it does not act on putrescine (Matsui and Pegg, 1980). There is an inverse correlation between the decrease in spermidine and putrescine acetylation which we observe at 8 hours and the peak of ODC (ornithine decarboxylase) activity reported by Pajunen et al. (1979). This inverse correlation suggests a mechanism by which polyamine levels are replenished. Such a hypothesis is supported by a concomitant increase in SAM-DC and GAD activities. Pajunen et al. (1979), were unable to detect differences in the formation of GABA from putrescine in control vs. allyglycine-treated mice. The use of 10 ~1 intraventricular injections in the mouse with distilled water as a vehicle as well as the large variations in specific activity, made such results hardly conclusive. Furthermore, this study (Pajunen et al., 1979) did not account for changes in polyamine metabolism, nor investigated formation of other possible polyamine metabolites, such as Y -glutamylputrescine or homocarnosine (Konishi et al., 1977).
Preliminary
TIME
Notes
AFTER
389
INJECTION
+...
PUTRESCINE
-+-
SPERMIDINE
(hr)
The actual formation of GABA from putrescine after inhibition of GAD, as well as the presence of acetylspermidine isomer(s) formed under such conditions, Furthermore, it is not known whether GABA derived remain to be investigated. from putrescine participates in neurotransmission. In conclusion, depletion of GABA levels, brought about by the administration of allylglycine, results in a biphasic change in the acetylation of putrescine and The initial increase in putrescine and spermidine acetylation spcrmidine. correlates well with a decrease in spermidine, spermine and GABA levels, on one hand, and with GAD and SAM-DC activities, on the other. The decrease in acetylation of putrescine and spermidine observed by us at 8 hours coincides with the peak of ODC activity and the return towards control values of GAD and SAMDC activities and of GABA and spermidine levels.
390
Preliminary
Notes
These results, as well as the presence of polyamine acetylation (Ortiz, Giacobini and Schmidt-Glenewinkel, 1983b) in the P2 and cytosolic fractions of the developing mouse brain, support the hypothesis of a contribution of putrescine to GABA levels during development, and possibly during periods of GABA depletion. ACKNOWLEDGEMENT J. G. 0. was a recipient of a predoctoral fellowship from the University of This work was supported by grants NS 11430 and NS 14086 to E. G. Connecticut.
REFERENCES BOLKENIUS, F. N. and SEILER, N. (1981) Acetyl derivatives in polyamine catabolism, Intl. J. Biochem. 13:287-292.
as intermediates
DEMELLO, F.G., BACHRACH, U. and NIREMBERG, M. (1976) Ornithine and glutamic acid decarboxylase activities in the developing chick retina, J. Neurochem. 27:847-851. KONISHI, H., NAKAJIMA, T. and SANO, I. (1977) Metabolism the central nervous system, J. Biochem. 81:355-360.
of putrescine
in
LIBBY, P.R. (1978) Calf-liver nuclear N-acetyltransferases: (purification and properties of two enzymes with both spermidine acetyltransferase and histone acetyltransferase activities), J. Biol. Chem. 235:233-237. MATSUI, I. and PEGG, A. E. (1980) Increase in the acetylation of spermidine in rat liver extracts brought about by treatment with carbon tetrachloride, Biochem. Biophys. Res. Commun. pp. 1009-1015. ORTIZ, J. G., GIACOBINI, E. and SCHMIDT-GLENEWINKEL, T. (1983a) Acetylation of polyamines in mouse brain: Subcellular and regional distribution, J. Neuroscience Res. 9:193-201. ORTIZ, J.G., GIACOBINI, E. and SCHMIDT-GLENEWINKEL, T. (198313) Polyamine acetylation in the developing and aging mouse brain, Intl. J. Developmental Neuroscience, 1: 179-185. PAJUNEN, A. E. I., HIETALA, O.A., BARUCH-VIRRANSALO, E. L. and PIHA, S. S. (1979) The effect of DL-allylglycine on polyamine and GABA metabolism in mouse brain, J. Neurochem. 32:1401-1408. SEILER, N. (1980) On the role of GABA in vertebrate Physiol. Chem. and Phys. 12:411-429.
polyamine metabolism,
SEILER, N., SCHMIDT-GLENEWINKEL, T. and SARHAN, S. (1979) On the formation of y -aminobutyric acid from putrescine in brain, J. Biochem. 86:277-278. SEILER, N. and SCHMIDT-GLENEWINKEL, T. (1975) Regional distribution of putrescine, spermidine and spermine in relation to the distribution of RNA and DNA in the rat nervous system, J. Neurochem. 24:791-795. SOBUE, K. and NAKAJIMA, T. (1978) Change in the concentrations of polyamines and Y -aminobutyric acid and their formation in chick embryo brain during development, J. Neurochem. 30:2’77-279.