Journul of Neurochemistry, 1977. Vol.
28, pp. 851-852. Pergamon Press. Printed in Great Britaln.
SHORT COMMUNICATION Effects of nerve growth factor and nerve growth factor antiserum on tyrosine hydroxylase and dihydropterine reductase activities in rat superior cervical ganglia in vim (Received 15 July 1976. Accepted 27 October 1976)
of tyrosine to DOPA is considered to be WILLIAMS et al. (19761, using 0.075 of the supernatant fracHYDROXKATION the rate-limiting step in the biosynthesis of catecholamines tion. Protein was determined in 0.05 ml of the supernatant (CA), and tyrosine hydroxylase (TH) the controlling portion, using the method of LOWRYet al. (1951). enzyme in this reaction (LEVITTet al., 1965). However, NGF, when administered to 5-day-old rats, produced MUSACCHIO et al. (1971) have suggested that the enzyme a sharp increase in the specific activity of TH in the SCG which recycles the cofactor for this reaction, dihydropteri- within 24 h after the injection (Table 1). However, the spedine reductase (DHPR), is rate-limiting under some condi- cific activity of DHPR was not changed at either 24 h or tions and that it may be an important factor in the deter- 48 h after the administration of NGF. The total activity mination of the in uiuo fates of CA-biosynthesis. For this of TH in control animals was 0.41 f 0.023 nmol of DOPA/ reason, as well as to obtain information about the action h/pair of SCG. The total activity of DHPR in control aniof nerve growth factor and the genetic regulation of T H mals was 101 f 4.1 nmol of NADH oxidized/5 min/pair of and DHPR, it was of interest to ask whether the changes SCG. Total protein per pair of ganglia in the controls was in TH activity in superior cervical ganglia (SCG) of rats 85 Ifr 4.7 p g . The administration of NGF caused no change after the administration of nerve growth factor (NGF) or after 24 h (83 & 4.9 pg), but 48 h after NGF the protein nerve growth factor antiserum (NGF-antiserum) (THOENEN content had increased to 106 4.5 pg per pair. In a second experiment NGF-antiserum was adminiset al., 1971; HENDRY& IVERSEN,1971) are accompanied tered subcutaneously to 5-day-old rats. The animals were by corresponding changes in the activity of DHPR. The 2.5 S nerve growth factor from the salivary glands killed 12, 24, or 48 h thereafter and the specific activities of adult male mice was prepared according to the method of TH and DHPR were measured (Table 2). There was a significant decrease in the specific activity of TH within & ANGELETTI (1969) and injected in a dose of BOCCHINI of 10 pg/g subcutaneously into 5-day-old rats. NGF-anti- 24 h. The activity of DHPR was not decreased in the first serum was made in sheep using a standard protocol and 24 h and only slightly between 24 and 48 h. Total protein was administered at a dose of Sop1 of a 10% aqueous per pair of ganglia in the controls was 66 & 2.7pg. The solution of the lyophilized antiserum per g into rats of protein content was essentially unchanged 12 h after the the same age. The animals were killed a t appropriate times antiserum (60 f 3.1 pg), but decreased substantially after by a blow to the head, and both SCG were removed under 24 (52 & 2.3 pg) and 48 h (41 f 1.8 pg). The results presented show that the alterations in TH a binocular dissecting microscope. One pair of SCG was homogenized in 0.3 ml of 5 mM-Tris buffer containing 0.1% activity in SCG, previously reported by others (THOENEN & IVERSEN, 1971), after the adminisTriton X-100 and centrifuged for 10min at 20,OOOg. TH et al., 1971; HENDRY activity was determined in 0.1 ml of the supernatant por- tration of NGF or NGF-antiserum are not accompanied tion, using the method of LEVITTet al. (1967) as modified by corresponding changes in the activity of DHPR. The by OESCHet al. (1973). DHPR activity was determined first statement which can be made from these data is that, by the method of NIELSENet al. (1969) as modified by under these conditions at least, the regeneration of reduced pteridines does not seem to be limiting for tyrosine hydroxylation. Indeed, the activity of DHPR is in enormous Abbreviations used: CA, catecholamines; DHPR, dihyd- excess in the ganglia compared even to the NGF-stimuropteridine reductase; NGF, nerve growth factor; SCG, lated activity of TH. A second conclusion which can be drawn regards the selective action of nerve growth factor superior cervical ganglia ; TH, tyrosine hydroxylase. NERVE GROWTH FACTOR (10 pg/g) ON THE SPECIFIC ACTIVITIES OF TYROSINE HYDROXYLASE AND DIHYDROPTERIDINE REDUCTASE IN SUPERIOR CERVICAL GANGLIA OF INFANT RATS
TABLE1. THE EFFECT OF 2.5 S
Tyrosine hydrox ylase* Time (h)
Control
NGF
Percentage of control
24 48
5.2 f 0.60 5.3 f 0.30
12.0 f 0.70$ 13.0 2 0.58#
231 245
* nm DOPA/h/mg protein (mean f s.E.M.). t nm NADH oxidized/5 min/mg protein (mean f s.E.M.). $ P < 0.005.
851
Dihydropteridine reductaset Percentage Control NGF of control
*
1040 50 1350 i.94
1100 f 36 1320 f 96
106 98
Short communication
852
TABLE 2. THEEFFECT OF NERVE GROWTH FACTOR-ANTISERUM (50 yl/g) ON THE
SPECIFIC ACTIVITIES OF TYROSINE HYDROXYLASE AND DIHYDROPTERIDINE REDUCTASE IN SUPERIOR CERVICAL GANGLIA OF INFANT RATS
Time (h) 12 24 48
Tyrosine hydroxylase* NGF Percentage of control Control an tiserum 9.1 _+ 0.68 7.6 & 1.1 8.5 0.14
10.5 & 1.7 3.6 k 0.789: 2.8 0.761
115 47 33
Dihydropteridine reductaset NGF Percentage Control an tiserum of control 1580 f 63 1430 & 94 1540 & 91
1910 & 745 1570 & 169 1230 k 111
121 110 80
* nm DOPA/h/mg protein (mean f s.E.M.). t nm NADH oxidized/5 min/mg protein (mean f s.E.M.). 3 P < 0.005. p P < 0.01.
and its antibody. The increase of TH activity after NGF administration is thought to be quite selective since other et al., 1971), now including related enzymes (THOENEN DHPR, do not rise. The action of the antibody must also be quite selective since enzymes related to TH, such as DHPR, do not decrease. And, finally, these data indicate that TH and DHPR are not coordinately regulated in mammals, although evidence for such linkage between DHPR and its relevant hydroxylase in bacteria has recently been presented (WILLIAMS et al., 1976).
REFERENCES
BOCCHINI V. & ANGELETTIP. U. (1969) Proc. natn. Acad. Sci., U.S.A. 64. 787-794. HENDRYI. A. & IVERSENL. L. (1971) Brain Res. 29, 159-1 62. LEVITTM., GIBBJ. W., DALYJ. W., LIPTONM. & UDENFRIEND S. (1967) Biochem. Pharmac. 16, 1313-1321. S., SJOERDSMA A. & UDENFRIEND S. LEVITTM., SPECTOR (1965) J . Pharmac. exp. Ther. 148, 1-8. LOWRY0. H., ROSEBROUGHN. J., FARRA. L. & RANDALL B. NIKODIJEVIC' R. J. (1951) J . biol. Chem. 193, 265-275. Section on Intermediary Metabolism, J. M., DANCELOG. L. & MCQUEENC. A. Lahoratory of Biomedical Sciences, M. W. Yu MUSACCHIO (1971) Proc. natn. Acad. Sci., U.S.A. 68, 2087-2091. G. GUROFF National Institute of Child Health V. & LIND K. E. (1969) Eur. NIELSENK. H., SIMONSEN and Human Development, J . Biochem. 9, 497-502. National Institutes of Health, OESCHF., OTTENU. & THOMENH. (1973) J . Neurochem. Bethesda, M D 20014, U.S.A. 20, 1691-1706. R. & THOENEN H., ANGELETTIP. U., LEVI-MONTALCINI KETTLER R. (1971) Proc. natn. Acad. Sci., U.S.A. 68, 1598-1602. WILLIAMS C. D., DICKENS G., LETENDRE C. H., GUROFF 'Present address: Department of Pharmacology, MediG., HAINESC. & SHIOTAT. (1976) J . Bacteriol. 127, 1197-1207. cal School, University of Skopje, Yugoslavia.
28, pp. 851-852. Pergamon Press. Printed in Great Britaln.
SHORT COMMUNICATION Effects of nerve growth factor and nerve growth factor antiserum on tyrosine hydroxylase and dihydropterine reductase activities in rat superior cervical ganglia in vim (Received 15 July 1976. Accepted 27 October 1976)
of tyrosine to DOPA is considered to be WILLIAMS et al. (19761, using 0.075 of the supernatant fracHYDROXKATION the rate-limiting step in the biosynthesis of catecholamines tion. Protein was determined in 0.05 ml of the supernatant (CA), and tyrosine hydroxylase (TH) the controlling portion, using the method of LOWRYet al. (1951). enzyme in this reaction (LEVITTet al., 1965). However, NGF, when administered to 5-day-old rats, produced MUSACCHIO et al. (1971) have suggested that the enzyme a sharp increase in the specific activity of TH in the SCG which recycles the cofactor for this reaction, dihydropteri- within 24 h after the injection (Table 1). However, the spedine reductase (DHPR), is rate-limiting under some condi- cific activity of DHPR was not changed at either 24 h or tions and that it may be an important factor in the deter- 48 h after the administration of NGF. The total activity mination of the in uiuo fates of CA-biosynthesis. For this of TH in control animals was 0.41 f 0.023 nmol of DOPA/ reason, as well as to obtain information about the action h/pair of SCG. The total activity of DHPR in control aniof nerve growth factor and the genetic regulation of T H mals was 101 f 4.1 nmol of NADH oxidized/5 min/pair of and DHPR, it was of interest to ask whether the changes SCG. Total protein per pair of ganglia in the controls was in TH activity in superior cervical ganglia (SCG) of rats 85 Ifr 4.7 p g . The administration of NGF caused no change after the administration of nerve growth factor (NGF) or after 24 h (83 & 4.9 pg), but 48 h after NGF the protein nerve growth factor antiserum (NGF-antiserum) (THOENEN content had increased to 106 4.5 pg per pair. In a second experiment NGF-antiserum was adminiset al., 1971; HENDRY& IVERSEN,1971) are accompanied tered subcutaneously to 5-day-old rats. The animals were by corresponding changes in the activity of DHPR. The 2.5 S nerve growth factor from the salivary glands killed 12, 24, or 48 h thereafter and the specific activities of adult male mice was prepared according to the method of TH and DHPR were measured (Table 2). There was a significant decrease in the specific activity of TH within & ANGELETTI (1969) and injected in a dose of BOCCHINI of 10 pg/g subcutaneously into 5-day-old rats. NGF-anti- 24 h. The activity of DHPR was not decreased in the first serum was made in sheep using a standard protocol and 24 h and only slightly between 24 and 48 h. Total protein was administered at a dose of Sop1 of a 10% aqueous per pair of ganglia in the controls was 66 & 2.7pg. The solution of the lyophilized antiserum per g into rats of protein content was essentially unchanged 12 h after the the same age. The animals were killed a t appropriate times antiserum (60 f 3.1 pg), but decreased substantially after by a blow to the head, and both SCG were removed under 24 (52 & 2.3 pg) and 48 h (41 f 1.8 pg). The results presented show that the alterations in TH a binocular dissecting microscope. One pair of SCG was homogenized in 0.3 ml of 5 mM-Tris buffer containing 0.1% activity in SCG, previously reported by others (THOENEN & IVERSEN, 1971), after the adminisTriton X-100 and centrifuged for 10min at 20,OOOg. TH et al., 1971; HENDRY activity was determined in 0.1 ml of the supernatant por- tration of NGF or NGF-antiserum are not accompanied tion, using the method of LEVITTet al. (1967) as modified by corresponding changes in the activity of DHPR. The by OESCHet al. (1973). DHPR activity was determined first statement which can be made from these data is that, by the method of NIELSENet al. (1969) as modified by under these conditions at least, the regeneration of reduced pteridines does not seem to be limiting for tyrosine hydroxylation. Indeed, the activity of DHPR is in enormous Abbreviations used: CA, catecholamines; DHPR, dihyd- excess in the ganglia compared even to the NGF-stimuropteridine reductase; NGF, nerve growth factor; SCG, lated activity of TH. A second conclusion which can be drawn regards the selective action of nerve growth factor superior cervical ganglia ; TH, tyrosine hydroxylase. NERVE GROWTH FACTOR (10 pg/g) ON THE SPECIFIC ACTIVITIES OF TYROSINE HYDROXYLASE AND DIHYDROPTERIDINE REDUCTASE IN SUPERIOR CERVICAL GANGLIA OF INFANT RATS
TABLE1. THE EFFECT OF 2.5 S
Tyrosine hydrox ylase* Time (h)
Control
NGF
Percentage of control
24 48
5.2 f 0.60 5.3 f 0.30
12.0 f 0.70$ 13.0 2 0.58#
231 245
* nm DOPA/h/mg protein (mean f s.E.M.). t nm NADH oxidized/5 min/mg protein (mean f s.E.M.). $ P < 0.005.
851
Dihydropteridine reductaset Percentage Control NGF of control
*
1040 50 1350 i.94
1100 f 36 1320 f 96
106 98
Short communication
852
TABLE 2. THEEFFECT OF NERVE GROWTH FACTOR-ANTISERUM (50 yl/g) ON THE
SPECIFIC ACTIVITIES OF TYROSINE HYDROXYLASE AND DIHYDROPTERIDINE REDUCTASE IN SUPERIOR CERVICAL GANGLIA OF INFANT RATS
Time (h) 12 24 48
Tyrosine hydroxylase* NGF Percentage of control Control an tiserum 9.1 _+ 0.68 7.6 & 1.1 8.5 0.14
10.5 & 1.7 3.6 k 0.789: 2.8 0.761
115 47 33
Dihydropteridine reductaset NGF Percentage Control an tiserum of control 1580 f 63 1430 & 94 1540 & 91
1910 & 745 1570 & 169 1230 k 111
121 110 80
* nm DOPA/h/mg protein (mean f s.E.M.). t nm NADH oxidized/5 min/mg protein (mean f s.E.M.). 3 P < 0.005. p P < 0.01.
and its antibody. The increase of TH activity after NGF administration is thought to be quite selective since other et al., 1971), now including related enzymes (THOENEN DHPR, do not rise. The action of the antibody must also be quite selective since enzymes related to TH, such as DHPR, do not decrease. And, finally, these data indicate that TH and DHPR are not coordinately regulated in mammals, although evidence for such linkage between DHPR and its relevant hydroxylase in bacteria has recently been presented (WILLIAMS et al., 1976).
REFERENCES
BOCCHINI V. & ANGELETTIP. U. (1969) Proc. natn. Acad. Sci., U.S.A. 64. 787-794. HENDRYI. A. & IVERSENL. L. (1971) Brain Res. 29, 159-1 62. LEVITTM., GIBBJ. W., DALYJ. W., LIPTONM. & UDENFRIEND S. (1967) Biochem. Pharmac. 16, 1313-1321. S., SJOERDSMA A. & UDENFRIEND S. LEVITTM., SPECTOR (1965) J . Pharmac. exp. Ther. 148, 1-8. LOWRY0. H., ROSEBROUGHN. J., FARRA. L. & RANDALL B. NIKODIJEVIC' R. J. (1951) J . biol. Chem. 193, 265-275. Section on Intermediary Metabolism, J. M., DANCELOG. L. & MCQUEENC. A. Lahoratory of Biomedical Sciences, M. W. Yu MUSACCHIO (1971) Proc. natn. Acad. Sci., U.S.A. 68, 2087-2091. G. GUROFF National Institute of Child Health V. & LIND K. E. (1969) Eur. NIELSENK. H., SIMONSEN and Human Development, J . Biochem. 9, 497-502. National Institutes of Health, OESCHF., OTTENU. & THOMENH. (1973) J . Neurochem. Bethesda, M D 20014, U.S.A. 20, 1691-1706. R. & THOENEN H., ANGELETTIP. U., LEVI-MONTALCINI KETTLER R. (1971) Proc. natn. Acad. Sci., U.S.A. 68, 1598-1602. WILLIAMS C. D., DICKENS G., LETENDRE C. H., GUROFF 'Present address: Department of Pharmacology, MediG., HAINESC. & SHIOTAT. (1976) J . Bacteriol. 127, 1197-1207. cal School, University of Skopje, Yugoslavia.
