Dopamine-s-Hydroxylase in the Rat Superior Cervical Ganglia

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 68, No. 7, pp , July 1971 Selective Induction by Nerve Growth Factor of Tyrosine Hydroxylase and Dopamine-s-Hydroxylase in the Rat Superior Cervical Ganglia (dopa decarboxylase/monoamine oxidase) H. THOENEN*, P. U. ANGELETTIt, R. LEVI-MIONTALCINIt, AND R. KETTLER* *Department of Experimental Pharmacology, Hoffman-La Roche, Basel, Switzerland; and tlaboratorio di Biologia Cellulare, C.N.R., Via Romagnosi 18A, Rome, Italy Contributed by R. Levi-Montalcini, May 6, 1971 ABSTRACT Treatment of newborn rats with 10 gg/g of nerve growth factor for 10 days enhanced not only the growth but also the differentiation of neuroblasts in superior cervical ganglia. These morphological changes were accompanied by selective induction of tyrosine hydroxylase and dopamine #-hydroxylase (EC ), whereas the total and specific activities of other enzymes involved in biosynthesis or metabolic degradation of norepinephrine rose only in proportion to the increase in volume of the sympathetic ganglia. There are remarkable similarities between this effect of nerve growth factor and the induction of trans-synaptic enzymes by increased activity of the sympathetic nervous system. Ever since the discovery that a specific nerve growth factor (NGF) enhances growth and differentiative processes of sympathetic neurons (1, 2), the question has been raised whether this factor selectively stimulates metabolic pathways characteristic of this particular nerve cell type. Sympathetic neurons offer an almost ideal system to test this hypothesis, since the biochemical correlates of their function are rather well known. However, it was not until recently that sufficiently sensitive methods became available to measure the activity of enzymes involved in the synthesis and metabolic degradation of the adrenergic neurotransmitter in small tissue samples (3-6). It thus became possible to study whether the hyperplastic and hypertrophic ganglia of newborn rats treated with NGF also differ from controls in the specific activity of these enzymes. The results to be reported here show that NGF, besides its characteristic morphological effects, also produces a selective induction of tyrosine hydroxylase and dopamine,3-hydroxylase (EC ), enzymes located in adrenergic neurons (7). In contrast, the activities of Dopa decarboxylase (EC ) and monoamine oxidase (EC ), enzymes which are present also in other cell types (7), increase only in proportion to the rise in volume of the adrenergic neurons in the superior cervical ganglia. METHODS Newborn rats (Wistar descent) of either sex were injected subcutaneously daily for 5 and 10 days with 10 ug/g of NGF dissolved in 0.9% NaCl. The NGF was prepared according to the method of Bocchini and Angeletti (8). The animals were killed by transection of the thorax above the heart. The superior cervical ganglia were removed under the dissecting Abbreviation: NGF, nerve growth factor microscope, fixed in Bouin's solution, and stained with toluidine blue. Ganglia of 5- and 10-day-old experimental and control littermates were then sectioned serially at 10,m. Cell counts were performed by inserting a micrometer disk into the ocular and counting all nerve cells in every other section of the experimental and control ganglia. For ultrastructural studies, the ganglia were fixed for 2 hr in 3% glutaraldehyde in 0.1 MI phosphate buffer (ph 7.4) with added CaCl2; the specimens were washed for 2 hr in the same buffer and postfixed in osmic hydroxide (1.33%) for 2 hr. Sections were cut with a Porter- Blum-Sorvall MT-2 microtome, stained with uranyl acetate in 50% ethanol or in lead hydroxide, and examined in a Philips 300 electron microscope. For the determination of tyrosine hydroxylase activity, each pair of ganglia was homogenized in 0.5 ml of ice-cold 0.25 M sucrose; for dopamine 0-hydroxylase, in 0.5 ml of M phosphate buffer (ph 7.5) containing 0.1% Triton X-100; for dopa decarboxylase, in 0.5 ml of 0.1 1\1 phosphate buffer (ph 7.0); and for monoamine oxidase in 0.1 M phosphate buffer ph 7.2. The activity of tyrosine hydroxylase was determined by the method of Levitt et al. (3), dopamine 0-hydroxylase according to Duch et al. (4), dopa decarboxylase according to Hakanson and Owman (5), with modifications described in detail by Thoenen et al. (9), and monoamine oxidase according to Wurtman and Axelrod (6). Enzyme activities are all expressed both in terms of product formed per hr per pair of ganglia (total activity) and in terms of product formed per hr per mg protein. The proteins were determined by the method of Lowry et al. (10). Enzyme kinetic parameters were determined by the method of Wilkinson (11) with a digital computer according to a program by Cleland (12). RESULTS Morphological effects of NGF on sympathetic ganglia in newborn rats The effects of daily injections of NGF in newborn rats for periods of 5 and 10 days are illustrated in Figs The TABLE 1. Cell number in superior cervical ganglia of 5-day-old NGF-treated and control rats Number of sections Cell number Control ,800 NGF-treated ,000

2 Proc. Nat. Acad. Sci. USA 68 (1971) Enzyme Induction by Nerve Growth Factor 1599 I X "t..1,. #Ij. A 1 E 41 FIGS Transverse sections through superior cervical ganglia of control (Fig. 1, X 114) and experimental (Fig. 2, X 114) animals (3-day-old rats), shown in whole mounts in Fig. 5 (X 13). Arrows in Fig. 5 indicate the level of sections. Figs. 3 and 4 (X 399) show size difference in sympathetic nerve cells of control (Fig. 3) and experimental (Fig. 4) ganglia reproduced in Figs. 1, 2, and 5. Note large fiber bundles among cells in NGF-treated ganglion. Figs. 6 and 7 (X855) show difference in size of sympathetic nerve cells of superior cervical ganglia of 10-day-old control (Fig. 6) and NGF-treated (Fig. 7) rats. volume difference of superior cervical ganglia of NGF-treated and control rats sacrificed at 5 days is shown in Fig. 5. Transverse sections of the same ganglia at low (Figs. 1 and 2) and high (Figs. 3 and 4) magnification give evidence for the size increase of sympathetic neurons in the ganglion of the experimental rat and for a marked difference in the texture of

3 1600 Proc. Nat. Acad. Sci. USA 68 (1971) Zoology: Thoenen et al. In: IL FIG.L8.iKS'Electron micrograph of cytoplasmic area of sympathetic neuron of 10-day-old rat treated since birth with NGF. Large neurofilament bundles fill part of the field. (X 13,940) ganglia. While in controls (Figs. 1 and 3) the nerve cell population is compact and evenly distributed, large fiber bundles segregate the population in the experimental ganglion into cell groups (Figs. 2 and 4). The total numbers of sections the and of nerve cells in these ganglia are given in Table 1. Figs. 6 and 7 show transverse sections of superior cervical ganglia of 10-day-old NGF-treated and control rats. The size increase of individual neurons is even more marked than in younger

4 Proc. Nat. Acad. Sci. USA 68 (1971) Enzyme Induction by Nerve Growth Factor 1601 TABLE 2. Effect of NGF on specific and total activities of enzymes in superior cervical ganglia of newborn rats Controls NGF-treated Specific Total Specific Total Tyrosine hydroxylase* 1.9 i± ± i i±0.02 DopamineB-hydroxylaset 11.8 ± ± i ± 0.6 Dopa decarboxylaset 0.32 ± ± ± ± 0.07 Monoamineoxidase 160- ± ± ±t ± 8.5 Activities (mean i SE, n = 6-8) are amounts of product formed per hr per mg protein (specific) and amounts of product formed per hr per pair of ganglia (total): * nanomoles of dopa, t picomoles of octopamine, t micromoles of dopamine, nanomoles of indoleacetic acid. TABLE 3. Kinetic characterization of tyrosine hydroxylase and in superior cervical ganglia of NGFtreated rats and controls dopamine jp-hydroxylase Tyrosine hydroxylase Km (MM tyrosine) Dopamine 0-hydroxylase Km (um tyramine) Controls 20.5 ± ± 34 NGF-treated 23.0 ± ±: 32 Newborn animals were treated for 10 days with 10 ug/g of NGF daily. specimens. The results of these studies, in agreement with those reported in mice (1, 2), indicate that the NGF calls forth hyperplastic and hypertrophic effects. Volume measurement of the superior cervical ganglia of 10-day-old experimental and control rats showed that the former is five times larger than the latter. On a protein basis, the average protein content per Total activity pair of ganglia amounts to 225 ± 10,g in the NGF-treated rat as compared to 65 i 2 ug in controls. Fig. 8 shows an electron micrograph of sympathetic nerve cells of the superior cervical ganglion of a rat treated with NGF from birth to the tenth day of age. The most impressive effect is the massive production of neurotubules and neurofilaments. This effect is in all respects similar to those elicited by NGF on sensory neurons in chick embryos in vitro and on sympathetic neurons in newborn mice (13, 14). Effect of NGF on total and specific enzyme activity in superior cervical ganglia Fig. 9 and Table 2 show that treatment of newborn rats for 10 days with NGF produced an 18-fold increase in total activity of tyrosine hydroxylase (product formed per hr per pair of ganglia) and a 13-fold increase in that of dopamine #-hydroxylase. The activities of dopa decarboxylase and monoamine oxidase rose only 4.5-fold and 2.5-fold, respectively. The Specific activity 2000 controls E I NGF 300 F 00 FIG. 9. Effect of NGF on enzymes involved in synthesis and metabolic degradation of norepinephrine. Newborn rats were treated with 10 pg/g of NGF for 10 days. The activity of all enzymes studied in the superior cervical ganglia is expressed in % of controls both for total (product formed per hr per pair of ganglia) and specific activity (product formed per hr per mg protein).

5 1602 Zoology: Thoenen et al. selectivity of the increase in enzyme activity becomes even clearer if one compares specific enzyme activity (product formed per hr per mg protein) of NGF-treated-animals with that of untreated controls. The ratio between the specific activity of NGF-treated animals and controls amounts to 5.4 for tyrosine hydroxylase, 3.6 for dopamine 0-hydroxylase, 1.5 for dopa decarboxylase, and 1.2 for monoamine oxidase (Fig. 4). The small increase in specific activity of the last two enzymes could be explained by a relative increase in the volume of neuronal versus satellite cells. In superior cervical ganglia of controls, the neuronal cells represent about 0.75 of the total volume. Mechanism of increased tyrosine hydroxylase and dopamine #-hydroxylase activity It could be assumed that the increased activity of these two enzymes results from a direct activation by NGF. To examine this possibility, we added 1-30 jg of NGF to enzyme preparations (100 al) used for the determination of tyrosine hydroxylase and dopamine #-hydroxylase activity. In both cases the enzyme activity remained unchanged after the in vitro addition of NGF. To determine whether the NGF promotes the formation of an activator, inhibits the production of an inhibitor, or enhances selectively the synthesis of tyrosine hydroxylase and dopamine,3-hydroxylase, we combined enzyme solutions from controls and NGF-treated animals in several proportions. Both for tyrosine hydroxylase and dopamine 0-hydroxylase, the activities of these combinations were always additive and therefore did not provide evidence for the loss of an inhibitor or appearance of an activator in the enzyme preparations of animals treated with NGF. Also, the fact that no significant change in Km values (Table 3) occurred speaks in favor of an increase in the amount of enzyme protein. DISCUSSION Evidence is here presented that NGF produces a selective 15- to 20-fold increase in the activity of tyrosine hydroxylase and dopamine,3-hydroxylase, enzymes that are exclusively located in adrenergic neurons (7). In contrast, the activity of dopa decarboxylase and monoamine oxidase rose only about in proportion to the increase in volume of sympathetic ganglia, corresponding to a 4-fold increase in their protein content. Proc. Nat. Acad. Sci. USA 68 (1971) The considerable rise in the activity of tyrosine hydroxylase and of dopamine,3-hydroxylase does not result from the formation of activators or disappearance of inhibitors, but rather from an increased synthesis of new enzyme protein We are therefore dealing with the selective induction of tvo0 enzymes that are exclusively located in adrenergic neurons by a specific growth factor. Enhanced activity of the peripheral sympathetic nervous system resulting from cold exposure, or from depletion of the adrenergic transmitter stores by reserpine produces, similar changes in the pattern of enzymes (15-17) involved in the synthesis of norepinephrine as does administration of NGp. This finding raises the question whether similar basic mechanisms are responsible in the two instances. It remains also to be elucidated whether this specific growth factor and the functional activity of neurons play similar roles in the growth regulation of the target cells during ontogenesis and in their adaptation to changed functional stages. 1. Levi-Montalcini, R., Harvey Lect., 60, 217 (1966). 2. Levi-Montalcini, R., and P. U. Angeletti, Physiol. Rev., 48, 534 (1968). 3. Levitt, M., J. WV. Gibb, J. WV. Daly, M. Lipton, and S. Udenfriend, Biochern. Pharmacol., 16, 1313 (1967). 4. Duch, D. S., 0. H. Viveros, and N. Kirshner, Biochern. Pharmacol., 17, 255 (1968). 5. HAkanson, R., and C. Owman, J. Neurochem., 13, 597 (1966). 6. Wurtman, R., and J. Axelrod, Biochem.. Pharmacol., 12, 1433 (1964). 7. Thoenen, H., "Catecholamines", in Handbook of Experimental Pharmacology (Springer, 1971), in press. 8. Bocchini, V., and P. U. Angeletti, Proc. Nat. Acad. Sci. USA, 64, 787 (1969). 9. Thoenen, H., R. Kettler, W. Burkard, and A. Saner, Naunyn-Schmiedebergs Arch. Pharmakol., in press. 10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Riot. Chem., 193, 265 (1951). 11. Wilkinson, G. N., Biochem. J., 80, 324 (1961). 12. Cleland, W. W., Nature, 198, 463 (1963). 13. Levi-Montalcini, R., F. Caramia, S. A. Luse, and P. U. Angeletti, Brain Res., 8, 347 (1968). 14. Angeletti, P. U., R. Levi-Montalcini, and F. Caramia, J. Ultrastruct. Res., 1971, in press. 15. Mueller, R. A., H. Thoenen, and J. Axelrod, J. Pharvmacol. Exp. Ther. 169, 74 (1969). 16. Mueller, R. A., H. Thoenen, and J. Axelrod, Mol. Pharmacol., 5, 463 (1969). 17. Molinoff, P. B., S. Brimijoin, R. Weinshilboum, and J. Axelrod, Proc. Nat. Acad. Sci. USA, 66, 453 (1970).