354
Bra& Research, 133 (1977) 354-357 © Elsevier/North-Holland Biomedical Press
Gas chromatographic-mass spectrometric assay of serotonin in rat superior cervical ganglia, Effects of nerve growth factor and 6-hydroxydopamine*
ANTONIA LIUZZI**, FREDRIK H. FOPPEN, JUAN M. SAAVEDRA, RITA LEVI-MONTALCINI and IRWIN J. KOPIN*** Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.) and (R.L.-M.) Laboratorio di Biologia Celhdare, Consiglio Nazionale delle Ricerche, Roma (Italy)
(Accepted June 2nd, 1977)
Nerve growth factor ( N G F ) isolated from mouse salivary gland as well as from other sources has striking effects on sympathetic neuronal growth 8. Histochemical studies have shown that there is a marked numerical increase in serotonin-like fluorescent cells in several tissues including the superior cervical ganglia and the iris of neonatal rats, when treated since birth with N G F 1. Although the presence of serotonin in sympathetic ganglia has been suggested by the demonstration in the ganglia of an enzyme that decarboxylates tryptophan 5, it is known that aromatic amino acid decarboxylase is a relatively non-specific enzyme 1°. Therefore the enzymatic activity could not be considered final proof" for the presence of this indoleamine. In the present study a highly specific gas chromatographic-mass spectrometric assay for serotonin has been used to determine its levels in rat superior cervical ganglia and irises. The biosynthetic enzyme 7 required for its formation was also measured. In experiments performed at the N.I.H., pregnant Sprague-Dawley rats (ZivicMiller, Allison Park, Pa.) were obtained on the lgth day of gestation and kept in individual cages until the litters were born. During the first three days of life the newborn pups were injected subcutaneously daily with 50 #1 saline containing bovine albumin (5/zg/g), N G F (5/zg/g) or 6-hydroxydopamine (50 #g/g) or both N G F and 6-hydroxydopamine. In another series of experiments, neonatal rats at the C.N.R. were injected with 10/zg/g N G F each day. After 4, 9 or 18 days the rats were killed by decapitation and the tissues were frozen immediately on dry ice and sent to the N.I.H. for analysis of serotonin. * A preliminary report was presented by Saavedra and Liuzzi at the Symposium on SIF Cells, N.I.H. (1976). ** On leave from the Laboratorio di Biologia Cellulare, C.N.R., Rome, Italy. *** To whom requests for Ieprints should be addressed.
355 The tissue parts were homogenized in 0.1 N HC1. To an aliquot of the homogenate 100 #10.1 N HC1, containing 2 ng serotonin-l, 1,2,2-D4 and 0.1 ~ (w/v) ascorbic acid were added. The supernatant was freeze-dried in a 5-ml round-bottom flask. Then 100 #1 pentafluoropropionic (PFP) anhydride and 10 #1 dry acetonitrile were added. After 30 min at room temperature the reaction mixture was blown to dryness in a nitrogen atmosphere. The reaction mixture was redissolved in 5 #1 acetonitrile and injected on a gas chromatography column, 150 cm × 2 ram, packed with 3 ~ OV-17 on Anakrom Q, 80-100 mesh. The column temperature was 220 °C and the helium flow was 20 ml/min. The retention time was 2.1 min. The gas chromatograph was connected with a Finnigan Model 1015C mass spectrometer provided with a programmable multiple ion monitor. The mass spectrometer was used under chemical ionization conditions with methane as reagent gas. The recording channels were preset on the amu-values of the most abundant ions of serotonin-di-PFP and serotonin-D4-di-PFP, 469 and 473. A standard curve was prepared adding known amounts of deuterated serotonin to ganglia and iris homogenates. Serotonin was also measured by a radiometric enzyme assaylL Tryptophanhydroxylase was measured according to Kizer et al. 7. N G F from mouse salivary glands was prepared according to Bocchini and AngelettiL Because of inconsistencies in recovery of iris tissue, results for this tissue were expressed in terms of proteins, measured according to Lowry et al. 11. Serotonin does not react with pentafluoropropionic anhydride in a uniform way. Because of the known differences in reactivity of the hydroxyl-group and the alkylamine-group in comparison with the NH-group of the indole ring, different reaction products could be expected; one, where only the OH- and NH2-groups have reacted, giving serotonin-di-PFP, and another one, with the OH-, NH2- and NH-groups substituted, giving serotonin-tri-PFP. Cattabeni et al. 4 have measured serotonin by means of the electron impact fragments of serotonin-tri-PFP. However, it is our experience that under various reaction conditions, including the one described by Cattabeni et al. 4, the main reaction product is always serotonin-di-PFP. Even under the most favorable conditions not more than 15 ~ of serotonin-tri-PFP was formed. A similar observation as to the reaction of pentafluoropropionic anhydride and tryptamine has been reported by Gelpi et al. 6. With such a loss of material it is hard to understand how Cattabeni et al. 4 could realize their stated degree of sensitivity. As could be expected of the more stable biogenic amines the chemical ionization spectra of the PFP-derivatives of serotonin showed their MH+-ions as their main fragments. In some of the experiments serotonin was also measured by an enzymatic-isotopic microassaylL The absolute amounts of serotonin were usually higher when measured by the gas chromatographic-mass spectrometric assay. This discrepancy diminished when the amount of serotonin per sample was higher. However, the relative difference in serotonin content between the two assays did not change significantly ~3. Differences in sample preparation and serotonin recovery might give an explanation. During development the serotonin level of the rat superior cervical ganglia increased from 0.9 ng/ganglion at birth to approximately 2.5 ng/ganglion at adulthood (Fig. 1). A significant elevation of the serotonin levels of the ganglia and also of the
356
2.5"
z 2.0-
t
_o,
z_ 1.5. z
1.0'
5
1'0
1'5
2'0
2'5
3'0
ADULT
AGE IN DAYS Fig. 1. Serotonin content of rat superior cervical ganglia during development. Serotonin was measured by a gas chromatographic-mass spectrometric assay. The results are mean values (4- S.E.M.) of 6 ganglia.
iris of 8-day-old rats was observed after t r e a t m e n t with N G F during the first three days of life (Table I). Similar increases were o b t a i n e d when n e w b o r n rats were treated daily with N G F for 4, 9 or 18 days (Table II). The tryptophan-hydroxylase levels o f the ganglia were also increased after t r e a t m e n t with N G F . T r e a t m e n t with 6 - h y d r o x y d o p a m i n e which destroys sympathetic nerves a n d nerve endings of i n f a n t rats °- lowered significantly the serotonin c o n c e n t r a t i o n in the iris. The decrease in serotonin in the ganglia, although measurable, was n o t significant.
TABLE I Serotonin and tryptophan-hydroxylase in superior cervical ganglia of newborn rats Newborn rats were treated with bovine albumin (5/~g/g), NGF (5 #g/g) and/or 6-hydroxydopamine (50 #g/g) since birth. They were killed after 8 days. Results are mean values (4- S.E.M.) for groups of 6-8 pairs of ganglia and 8-16 pairs of irises. Tryptophan-hydroxylase activity units are in pmoles/h. Treatment
Albumin NGF 6-Hydroxydopamine NGF 4- 6-hydroxydopamine
* P < 0.01.
Ganglia
Iris
Serotonin (ng/mg protein)
Tryptophanhydroxylase (units/ganglion)
Serotonin (ng/mg protein)
1.8 3.2 2.1 2.7
91 216 70 252
8.8 4- 1.1 19.1 ± 1.9" 3.8 4- 0.6 16.7 ± 1.8"
4- 0.3 4- 0.4* ± 0.2 4- 0.2*
± 13 4- 46* 4-9 4- 60*
357 TABLE II Effect of NGF treatment on serotonin in rat superior cervical ganglia
Newborn rats were treated with N G F (10 #g/g) daily since birth for 4, 9 or 18 days. Serotonin levels in superior cervical ganglia were compared with control animals. Values are means (4- S.E.M.) of 8 pairs of ganglia. Treatment (days)
Serotonin in NGF-treated ganglia vs. control ganglia (%)
4 9 18
255 4- 28 180 :k 25 345 =k 22
C o m b i n e d t r e a t m e n t with N G F a n d 6 - h y d r o x y d o p a m i n e has effects on serotonin a n d t r y p t o p h a n - h y d r o x y l a s e which were similar to those o f N G F alone (Table 1). The results o f the present study confirm the histological observations I t h a t t r e a t m e n t with N G F increases the serotonin contents o f the superior cervical ganglia a n d the iris. The parallel increase in t r y p t o p h a n - h y d r o x y l a s e o f the ganglia suggests that serotonin is synthesized in the ganglia a n d t h a t it is n o t the result o f infiltration with platelets which c o n t a i n serotonin b u t n o t t r y p t o p h a n - h y d r o x y l a s e 9. Therefore it a p p e a r s t h a t N G F stimulation o f serotonin f o r m a t i o n in the ganglia is due to action on cells t h a t are present in the ganglia. Since 6 - h y d r o x y d o p a m i n e t r e a t m e n t lowered serotonin c o n t e n t o f the iris, b u t n o t o f the ganglia, it is possible t h a t the toxic a m i n e is a c c u m u l a t e d in these s e r o t o n i n - c o n t a i n i n g cells, p a r t i c u l a r l y in the iris. The toxic effect o f the amine, however, does n o t a p p e a r to d e s t r o y these cells, as N G F is still able to p r o d u c e a highly significant increase in serotonin, even in the presence o f 6h y d r o x y d o p a m i n e . The n a t u r e o f the N G F - s t i m u l a t e d s e r o t o n i n - c o n t a i n i n g cells r e m a i n s u n k n o w n , b u t it is unlikely t h a t they are s y m p a t h e t i c neurons. 1 Aloe, L. and Levi-Montalcini, R., Mast cells increase in tissue of neonatal rats injected with nerve growth factor, Brain Research, 133 (1977) 358-366. 2 Ange•etti•P.U.•Chemica•sympathect•myinnewb•rnanima•s•Neur•pharmac•••gy•••(•97•)55-59.
3 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000 molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787-794. 4 Cattabeni, F., Koslow, S. H. and Costa, E., Gas chromatographic-mass spectrometric assay of four indole alkylamines of rat pineal, Science, 178 (1972) 166-168. 5 Gaddum, J. H. and Giarman, N. J., Preliminary studies on the biosynthesis of 5-hydroxytlyptamine, Brit. J. Pharmacol. Chemother., 11 (1956) 88-92. 6 Gelpi, E., Peralta, E. and Segura, J., Gas chromatography-mass spectrometry of catecholamines and tryptamines, J. Chromatog. Sci., 12 (1974) 701-709. 7 Kizer, S., Zivin, J.,Saavedra, J. M. and Brownstein, M., A sensitive immunoassay for tryptophanhydroxylase in brain, J. Neurochem., 24 (1975) 779-785. 8 Levi-Montalcini, R., The nerve growth factor: its mode of action on sensory and sympathetic nerve cells, Harvey Lect. Set., 60 (1966) 217-259. 9 Lovenberg, W., personal communication. 10 Lovenberg, W., Weissbach, H. and Udenfriend, S., Aromatic L-amino acid decarboxylase, J. bioL Chem., 237 (1962) 89-93. 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measmement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 12 Saavedra, J. M., Brownstein, M. and Axelrod, J., A specific and sensitive enzymatic-isotopic microassay for serotonin in tissue, J. PharmacoL exp. Ther., 186 (1973) 508-515. 13 Saavedra, J. M. and Liuzzi, A., Nerve Growth Factor: effects on 5-hydroxytryptamine and phenylethanolamine-N-methyltransferase in the superior cervical ganglia of the rat, In O. Erankti (Ed.), SIF Cells, Fogarty International Center Proceeding VoL 30, 1976, pp. 124-130.
Bra& Research, 133 (1977) 354-357 © Elsevier/North-Holland Biomedical Press
Gas chromatographic-mass spectrometric assay of serotonin in rat superior cervical ganglia, Effects of nerve growth factor and 6-hydroxydopamine*
ANTONIA LIUZZI**, FREDRIK H. FOPPEN, JUAN M. SAAVEDRA, RITA LEVI-MONTALCINI and IRWIN J. KOPIN*** Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.) and (R.L.-M.) Laboratorio di Biologia Celhdare, Consiglio Nazionale delle Ricerche, Roma (Italy)
(Accepted June 2nd, 1977)
Nerve growth factor ( N G F ) isolated from mouse salivary gland as well as from other sources has striking effects on sympathetic neuronal growth 8. Histochemical studies have shown that there is a marked numerical increase in serotonin-like fluorescent cells in several tissues including the superior cervical ganglia and the iris of neonatal rats, when treated since birth with N G F 1. Although the presence of serotonin in sympathetic ganglia has been suggested by the demonstration in the ganglia of an enzyme that decarboxylates tryptophan 5, it is known that aromatic amino acid decarboxylase is a relatively non-specific enzyme 1°. Therefore the enzymatic activity could not be considered final proof" for the presence of this indoleamine. In the present study a highly specific gas chromatographic-mass spectrometric assay for serotonin has been used to determine its levels in rat superior cervical ganglia and irises. The biosynthetic enzyme 7 required for its formation was also measured. In experiments performed at the N.I.H., pregnant Sprague-Dawley rats (ZivicMiller, Allison Park, Pa.) were obtained on the lgth day of gestation and kept in individual cages until the litters were born. During the first three days of life the newborn pups were injected subcutaneously daily with 50 #1 saline containing bovine albumin (5/zg/g), N G F (5/zg/g) or 6-hydroxydopamine (50 #g/g) or both N G F and 6-hydroxydopamine. In another series of experiments, neonatal rats at the C.N.R. were injected with 10/zg/g N G F each day. After 4, 9 or 18 days the rats were killed by decapitation and the tissues were frozen immediately on dry ice and sent to the N.I.H. for analysis of serotonin. * A preliminary report was presented by Saavedra and Liuzzi at the Symposium on SIF Cells, N.I.H. (1976). ** On leave from the Laboratorio di Biologia Cellulare, C.N.R., Rome, Italy. *** To whom requests for Ieprints should be addressed.
355 The tissue parts were homogenized in 0.1 N HC1. To an aliquot of the homogenate 100 #10.1 N HC1, containing 2 ng serotonin-l, 1,2,2-D4 and 0.1 ~ (w/v) ascorbic acid were added. The supernatant was freeze-dried in a 5-ml round-bottom flask. Then 100 #1 pentafluoropropionic (PFP) anhydride and 10 #1 dry acetonitrile were added. After 30 min at room temperature the reaction mixture was blown to dryness in a nitrogen atmosphere. The reaction mixture was redissolved in 5 #1 acetonitrile and injected on a gas chromatography column, 150 cm × 2 ram, packed with 3 ~ OV-17 on Anakrom Q, 80-100 mesh. The column temperature was 220 °C and the helium flow was 20 ml/min. The retention time was 2.1 min. The gas chromatograph was connected with a Finnigan Model 1015C mass spectrometer provided with a programmable multiple ion monitor. The mass spectrometer was used under chemical ionization conditions with methane as reagent gas. The recording channels were preset on the amu-values of the most abundant ions of serotonin-di-PFP and serotonin-D4-di-PFP, 469 and 473. A standard curve was prepared adding known amounts of deuterated serotonin to ganglia and iris homogenates. Serotonin was also measured by a radiometric enzyme assaylL Tryptophanhydroxylase was measured according to Kizer et al. 7. N G F from mouse salivary glands was prepared according to Bocchini and AngelettiL Because of inconsistencies in recovery of iris tissue, results for this tissue were expressed in terms of proteins, measured according to Lowry et al. 11. Serotonin does not react with pentafluoropropionic anhydride in a uniform way. Because of the known differences in reactivity of the hydroxyl-group and the alkylamine-group in comparison with the NH-group of the indole ring, different reaction products could be expected; one, where only the OH- and NH2-groups have reacted, giving serotonin-di-PFP, and another one, with the OH-, NH2- and NH-groups substituted, giving serotonin-tri-PFP. Cattabeni et al. 4 have measured serotonin by means of the electron impact fragments of serotonin-tri-PFP. However, it is our experience that under various reaction conditions, including the one described by Cattabeni et al. 4, the main reaction product is always serotonin-di-PFP. Even under the most favorable conditions not more than 15 ~ of serotonin-tri-PFP was formed. A similar observation as to the reaction of pentafluoropropionic anhydride and tryptamine has been reported by Gelpi et al. 6. With such a loss of material it is hard to understand how Cattabeni et al. 4 could realize their stated degree of sensitivity. As could be expected of the more stable biogenic amines the chemical ionization spectra of the PFP-derivatives of serotonin showed their MH+-ions as their main fragments. In some of the experiments serotonin was also measured by an enzymatic-isotopic microassaylL The absolute amounts of serotonin were usually higher when measured by the gas chromatographic-mass spectrometric assay. This discrepancy diminished when the amount of serotonin per sample was higher. However, the relative difference in serotonin content between the two assays did not change significantly ~3. Differences in sample preparation and serotonin recovery might give an explanation. During development the serotonin level of the rat superior cervical ganglia increased from 0.9 ng/ganglion at birth to approximately 2.5 ng/ganglion at adulthood (Fig. 1). A significant elevation of the serotonin levels of the ganglia and also of the
356
2.5"
z 2.0-
t
_o,
z_ 1.5. z
1.0'
5
1'0
1'5
2'0
2'5
3'0
ADULT
AGE IN DAYS Fig. 1. Serotonin content of rat superior cervical ganglia during development. Serotonin was measured by a gas chromatographic-mass spectrometric assay. The results are mean values (4- S.E.M.) of 6 ganglia.
iris of 8-day-old rats was observed after t r e a t m e n t with N G F during the first three days of life (Table I). Similar increases were o b t a i n e d when n e w b o r n rats were treated daily with N G F for 4, 9 or 18 days (Table II). The tryptophan-hydroxylase levels o f the ganglia were also increased after t r e a t m e n t with N G F . T r e a t m e n t with 6 - h y d r o x y d o p a m i n e which destroys sympathetic nerves a n d nerve endings of i n f a n t rats °- lowered significantly the serotonin c o n c e n t r a t i o n in the iris. The decrease in serotonin in the ganglia, although measurable, was n o t significant.
TABLE I Serotonin and tryptophan-hydroxylase in superior cervical ganglia of newborn rats Newborn rats were treated with bovine albumin (5/~g/g), NGF (5 #g/g) and/or 6-hydroxydopamine (50 #g/g) since birth. They were killed after 8 days. Results are mean values (4- S.E.M.) for groups of 6-8 pairs of ganglia and 8-16 pairs of irises. Tryptophan-hydroxylase activity units are in pmoles/h. Treatment
Albumin NGF 6-Hydroxydopamine NGF 4- 6-hydroxydopamine
* P < 0.01.
Ganglia
Iris
Serotonin (ng/mg protein)
Tryptophanhydroxylase (units/ganglion)
Serotonin (ng/mg protein)
1.8 3.2 2.1 2.7
91 216 70 252
8.8 4- 1.1 19.1 ± 1.9" 3.8 4- 0.6 16.7 ± 1.8"
4- 0.3 4- 0.4* ± 0.2 4- 0.2*
± 13 4- 46* 4-9 4- 60*
357 TABLE II Effect of NGF treatment on serotonin in rat superior cervical ganglia
Newborn rats were treated with N G F (10 #g/g) daily since birth for 4, 9 or 18 days. Serotonin levels in superior cervical ganglia were compared with control animals. Values are means (4- S.E.M.) of 8 pairs of ganglia. Treatment (days)
Serotonin in NGF-treated ganglia vs. control ganglia (%)
4 9 18
255 4- 28 180 :k 25 345 =k 22
C o m b i n e d t r e a t m e n t with N G F a n d 6 - h y d r o x y d o p a m i n e has effects on serotonin a n d t r y p t o p h a n - h y d r o x y l a s e which were similar to those o f N G F alone (Table 1). The results o f the present study confirm the histological observations I t h a t t r e a t m e n t with N G F increases the serotonin contents o f the superior cervical ganglia a n d the iris. The parallel increase in t r y p t o p h a n - h y d r o x y l a s e o f the ganglia suggests that serotonin is synthesized in the ganglia a n d t h a t it is n o t the result o f infiltration with platelets which c o n t a i n serotonin b u t n o t t r y p t o p h a n - h y d r o x y l a s e 9. Therefore it a p p e a r s t h a t N G F stimulation o f serotonin f o r m a t i o n in the ganglia is due to action on cells t h a t are present in the ganglia. Since 6 - h y d r o x y d o p a m i n e t r e a t m e n t lowered serotonin c o n t e n t o f the iris, b u t n o t o f the ganglia, it is possible t h a t the toxic a m i n e is a c c u m u l a t e d in these s e r o t o n i n - c o n t a i n i n g cells, p a r t i c u l a r l y in the iris. The toxic effect o f the amine, however, does n o t a p p e a r to d e s t r o y these cells, as N G F is still able to p r o d u c e a highly significant increase in serotonin, even in the presence o f 6h y d r o x y d o p a m i n e . The n a t u r e o f the N G F - s t i m u l a t e d s e r o t o n i n - c o n t a i n i n g cells r e m a i n s u n k n o w n , b u t it is unlikely t h a t they are s y m p a t h e t i c neurons. 1 Aloe, L. and Levi-Montalcini, R., Mast cells increase in tissue of neonatal rats injected with nerve growth factor, Brain Research, 133 (1977) 358-366. 2 Ange•etti•P.U.•Chemica•sympathect•myinnewb•rnanima•s•Neur•pharmac•••gy•••(•97•)55-59.
3 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000 molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787-794. 4 Cattabeni, F., Koslow, S. H. and Costa, E., Gas chromatographic-mass spectrometric assay of four indole alkylamines of rat pineal, Science, 178 (1972) 166-168. 5 Gaddum, J. H. and Giarman, N. J., Preliminary studies on the biosynthesis of 5-hydroxytlyptamine, Brit. J. Pharmacol. Chemother., 11 (1956) 88-92. 6 Gelpi, E., Peralta, E. and Segura, J., Gas chromatography-mass spectrometry of catecholamines and tryptamines, J. Chromatog. Sci., 12 (1974) 701-709. 7 Kizer, S., Zivin, J.,Saavedra, J. M. and Brownstein, M., A sensitive immunoassay for tryptophanhydroxylase in brain, J. Neurochem., 24 (1975) 779-785. 8 Levi-Montalcini, R., The nerve growth factor: its mode of action on sensory and sympathetic nerve cells, Harvey Lect. Set., 60 (1966) 217-259. 9 Lovenberg, W., personal communication. 10 Lovenberg, W., Weissbach, H. and Udenfriend, S., Aromatic L-amino acid decarboxylase, J. bioL Chem., 237 (1962) 89-93. 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measmement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 12 Saavedra, J. M., Brownstein, M. and Axelrod, J., A specific and sensitive enzymatic-isotopic microassay for serotonin in tissue, J. PharmacoL exp. Ther., 186 (1973) 508-515. 13 Saavedra, J. M. and Liuzzi, A., Nerve Growth Factor: effects on 5-hydroxytryptamine and phenylethanolamine-N-methyltransferase in the superior cervical ganglia of the rat, In O. Erankti (Ed.), SIF Cells, Fogarty International Center Proceeding VoL 30, 1976, pp. 124-130.
