Comparison of Tyrosine Hydroxylase and Choline Acetyltransferase Activity in Response to Sympathetic Nervous System Activation

192 Brain Research, 156 (1978) 192-197 ,~ Elsevier/North-Holland Biomedical Press

Comparison of tyrosine hydroxylase and choline acetyltransferase activity in response to sympathetic nervous system activation

DONALD D. LUND, MARK M. KNUEPFER, MICHAEL J. BRODY, RANBIR K. BHAT- NAGAR, PHILLIP G. SCHMID and ROBERT ROSKOSKI, JR. Departments of Biochemistry, Pharmacology, Internal Medicine and The Cardiovascular Center, The University of Iowa, Iowa City, Iowa 52242 ( U.S.A. ) (Accepted June 15th, 1978)

Choline acetyltransferase and tyrosine hydroxylase serve as enzyme markers for preganglionic and postganglionic sympathetic nerves, respectively. There is abundant evidence that experimental conditions which increase the activity of the peripheral sympathetic nervous system produce an increase in the activity of neuronal tyrosine hydroxylase and dopamine fl-hydroxylase 11. The activity of these enzymes serve as indices of sympathetic neural activity. It is not known, however, if choline acetyltransferase activity also serves as a reliable index of neural activity. Initial studies by Mueller and co-workers showed that 6-hydroxydopamine t4 and reserpine injection 15 increased the activity of tyrosine hydroxylase within the terminals of adrenergic neurons and in the adrenal medulla. Molinoff et al. 12 demonstrated a neurally mediated increase in dopamine-fl-hydroxylase. Subsequent studies employing a variety of conditions such as cold 1,2a, immobilization 8, amphetamine 10, insulin 25, phenoxybenzamine a, swimming 24 and hypothalamic stimulation is also increase tyrosine hydroxylase activity in the peripheral sympathetic system and adrenals. Since the increase in tyrosine hydroxylase activity is blocked by RNA and protein synthesis inhibitors 16, it was suggested that there is an increase in enzyme synthesis. Using immunoprecipitation, Chuang and Costa 1 showed that there is an increase in enzyme protein in response to cold stress in rats. In addition to the increased activity requiring protein synthesis, there may be a short-term regulation of activity, associated with protein phosphorylation 7,~ and changes in apparent substrate affinities 26. In contrast to tyrosine hydroxylase, relatively few studies have been performed on the regulation of choline acetyltransferase activity in the sympathetic nervous system. Oesch has, for example, examined the effect of reserpine injection into rats on the activity of choline acetyltransferase in stellate and superior cervical ganglia and in the adrenal medulla 17. He reported that the activity increases in response to reserpine treatment. As reported in preliminary form 2~, we find that tyrosine hydroxylase activity is more responsive than choline acetyltransferase and that large, perhaps supraphy- siologic, stimuli are necessary to increase activity. 193

Male Sprague-Dawley rats (100-120 g or 200-250 g, as specified) were obtained from Biolabs (St. Paul, Minn.) and housed in groups of 6 in a controlled (24 °C) environment. The animals were exposed to light 12 h per day and allowed food and water ad lib±turn. To allow for acclimatization, no experimental manipulations were performed until one week after the animals were received. After the specified treatment, the animals were sacrificed by a blow to the neck unless otherwise noted. The superior cervical and stellate ganglia and the adrenal medulla were removed, weighed, placed in polyethylene vials and frozen in liquid N2 as rapidly as possible (within 10 min) and stored. After storage, the tissues were homogenized in 0.3 ml of ice-cold 5 mM potassium phosphate (pH 7.4) containing 0.2 ~ Triton X-100 with a glass-to-glass Duall tissue grinder (Kontes; Vineland, N.J.). Duplicate portions (10/~l) were taken for choline acetyltransferase measurements by the procedure of Roskoski tg. After the remainder was centrifuged at 13,000 × g (5 rain, 4 °C) in an Eppendorf microfuge, tyrosine hydroxylase activity was measured in 50/~I portions of the supernatant by the method of Coyle 2 using 0.2 mM tyros±he and 0.9 m M DM PH4. At these concentrations, measured activity therefore approximates the Vm~x; observed changes in activity are not due to alterations in the Km of the enzyme for substrate or cofactor. ~6 Student's 't'-test was used to establish significance of differences between means. A P < 0.05 was chosen as the level of significance. In agreement with other investigators, we find that reserpine injection (7.5 mg/kg) produces an increase in tyrosine hydroxylase activity in the adrenal gland and stellate ganglia within 24 h (Table I). After injection of this large dose, the animals

TABLE i

Effect of reserpine on tyros±he hydroxylase (TH) and choline acetyltran~ferase (CA T) activity in 6Tm pathetic ganglia and adrenal medulla Reserpine (7.5 mg/kg) was injected intraperitoneally into 6 rats (100-120 g); the 6 control rats received on ly vehicle. After 24 h, the specified tissue was removed and stored prior to assay. Aliquots of the whole homogenate were taken for choline acetyltransferase measurement (CAT). Portions of the homogenate were centrifuged (27,000 × g, 10 rain) and transferase activity was determined in the supernatant fraction (CAT Sup). Tyrosine hydroxylase was measured as described in the text. Results are expressed as nmole/mg protein/h.

Superior Stellate ganglia Adrenal medulla cervical ganglia

CA T CA T CA T Sup TH CA T CA T Sup TH

Control 91.5 90.6 11.4 66.5 4.26 0.96 45.8 i: 5.7 ± 10.8 ± 4.4 ± 12.5 ± 0.42 ± 0.18 ± 3.1 Reserpine 94.4 96.0 13.9 102.0" 4.56 1.80 97.8* ± 7.2 ± 9.6 ± 6.0 5< 10.3 ± 0.30 ± 0.36 ± 8.5

* Significantly different from control (P < 0.05). 194 develop ptosis, tearing, diarrhea, and become quiescent. In contrast to Oesch ~7, however, we did not observe an increase in choline acetyltransferase activity in superior cervical or stellate ganglia or adrenal medulla (Table I). We also obtained similar results two days after reserpine injection. The 24 h experiment was performed 3 times with animals weighing 200-250 g and 3 times with animals weighing 100-120 g. Experiments were then performed to ascertain the difference between our findings and those of Oesch 17. In contrast to the latter investigator, who measured choline acetyltransferase activity in the 27,000 × g supernatant in a Triton X-100 extract, we routinely measure activity in the whole homogenate 2°. As noted by Oesch aT, we find about 25 ~ of enzyme activity occurs in the supernatant. He also notes that the increases observed did not consistently reach statistical significance using whole homogenate. However, we have been unable to detect a statistically significant increase. Following the methodology of Fonnum 6, experiments were performed to ascertain the means by which the choline acetyltransferase binds to the 27,000 x g membrane fraction. On the one hand, Triton X-100 fails to solubilize the transferase; on the other hand, 0.2 M KC1 completely solubilizes the enzyme (not shown). NaCI (0.2 M) also renders the enzyme soluble. We conclude, like Fonnum, that the enzyme is bound to the membrane fractions by electrostatic forces. We also conclude that reserpine injection fails to increase the total choline acetyltransferase activity at 24 (Table I) and 48 h in the adrenal or the stellate and superior cervical sympathetic ganglia. To follow up the reserpine studies where a consistent dissociation between choline acetyltransferase and tyrosine hydroxylase activity was observed, a second method -- cold exposure -- was used to increase the activity of the sympathetic system. Thoenen 2a and Chuang and Costa 1 have shown that maintaining rats at 4 °C for as little as 2 h produced an increase in adrenal tyrosine hydroxylase activity after 1-2 days. In our experiments, individually caged rats (100-120 g) were kept in a 4 °C cold room for 8 days. The control animals were also individually caged. Under these conditions, we find no change in superior cervical or stellate ganglion nor in adrenal medullary choline acetyltransferase activity. TH activity, however, was significantly increased in each tissue (not shown). Choosing a third means for activating the sympathetic system, electrical stimulation of the posterior hypothalamus was performed as described by Folkow and Rubinstein 5. One group of rats was stimulated for 24 h and a second group for 48 h. At the time the stimulator was on, the rats exhibited a typical alerting and avoidance response. They usually tried to escape from the cage each time they were stimulated. Each stimulation also increased arterial pressure. During the 24 or 48 h period of stimulation, current was increased as needed in order to maintain the blood pressure increase at a constant level. Individual pressure increments averaged 24 _4- I mm Hg in the 6 stimulated rats. Resting mean arterial pressure, however, was not significantly altered by 48 h of stimulation, averaging 120 :k 2 mm Hg at beginning and 124 ~ 4 mm Hg at the end of the experiment. In addition, no change was noted in the heart rate during stimulation (396 ~ 14 beats/rain) when compared to the control period 195

(394 ± 7 beats/rain). The pressure responses produced by hypothalamic stimulation appear to be produced by selective activation of sympathetic nerves to cardiovascular effectors and not by release of adrenal medullary catecholamines. Thus, responses were not altered by adrenal demedullation but were reduced significantly by the ganglionic blocker hexamethonium (not shown). After 24 h of stimulation, there was no change in choline acetyltransferase or tyrosine hydroxylase activity in the tissues sampled. Even after 48 h of stimulation, tyrosine hydroxylase activity was significantly elevated only in the adrenal medulla (Table II). It was unchanged in the stellate and superior cervical ganglia. Again, we did not observe any increases in choline acetyltransferase activity. The activity of enzymes mediating neurotransmitter metabolism has been used by many investigators as an index of neuronal activity. We wished to ascertain the relative sensitivity of choline acetyltransferase and tyrosine hydroxylase, respective marker enzymes of the preganglionic and postganglionic sympathetic nervous system, in response to sympathetic nerve activation. We used 3 experimental approaches to activate the sympathetic system: reserpine injection, cold exposure, and electrical stimulation of the hypothalamus. The results were consistent and readily reproducible, and indicate that tyrosine hydroxylase activity is a more sensitive indicator of sympathetic activity than choline acetyltransferase. Using similar methods, we also found that adrenal tyrosine hydroxylase activity increases in guinea pigs in response to cold exposure under conditions where choline acetyltransferase activity remains unchanged (not shown). This suggests that the finding is not species-specific. An increase of blood pressure during hypothalamic stimulation indicates

TABLE 11 Effect of hypothalamic stimulation on tyrosine hydroxylase (TH) and choline acetyltransferase (CAT) activity in ,sTmpathetic ganglia and adrenal medulla Electrodes were implanted into 6 experimental and 6 control animals (200-250 g) after Nembutal anesthesia (50 mg/kg) and atropine (20/~g/kg). A cannula was inserted in the lower aorta, and a bipolar electrode was implanted in the posterior hypothalamus7 using a Kopf stereotaxic apparatus (0.3 mm posterior to bregma, 1.0 mm lateral to bregma and 8.0 mm below the dura) 0. After a 48 h recovery the cannula was attached to a Statham P23 AA transducer to monitor arterial blood pressure. A Grass $48 Stimulator and a Grass CCUI constant current unit were used to deliver square wave 45-200/tA trains ( 1 msec every 10 msec) for 5 sec every 30 sec for 24 h and 10 sec every 30 sec from 25 through 48 h via freely moving swivels to the electrode. Sham animals were similarly attached but did not receive any current. After 48 h of stimulation, the animals were anesthetized with Nembutal (50 mg/kg) and sacrificed prior to tissue sampling. Enzyme activity is expressed as nmole/mg protein/h.

Superior cervical Stellate ganglia Adrenal medulla ganglia CAT TH CAT TH CAT TH

Control 45.8 74.1 27.6 81.0 3.72 28.1 -- 6.3 ± 6.5 ~ 1.6 ~ 2.7 ± 0.42 ± 3.9 48 h Stimulation 54.3 ± 75.4 37.7 89.8 3.00 43.3* ± 3.8 ± 18.2 ± 4.5 £ 12.7 ± 0.24 i: 4.3

* Significantly different from control (P < 0.05). 196 sympathetic neural system activation. The degree of this activation, however, is diffi- cult to quantify. We consider the hypothalamic stimulation (10-20 sec/min for 24-48 h), which prompts these blood pressure and alerting responses and maintains the animal in an active state for 48 continuous hours, to be a rather potent sympathetic stimulus. We anticipated that this intervention would elevate tyrosine hydroxylase activity in ganglia and the adrenal. Since elevation was found only in the adrenal after 48 h stimulation, we feel that rather marked stimulation is required to elicit this response. With reserpine injection, the dosage routinely employed (7.5 mg/kg) incapacitates the animals. The exposure of small animals to cold also produces a marked increase in sympathetic activity 1,23. In contrast to Oesch 17, we were unable to demonstrate an increase in choline acetyltransferase activity in response to reserpine. One difference in methodology partially accounts for the discrepancy. We measured activity in whole homogenate and Oesch measured activity in the Triton X-100 solubilized 27,000 /, g supernatant fraction. We were unable to show a change in this fraction, even though there was a measurable increase in tyrosine hydroxylase activity. In closer agreement with our results, he states that, when activity of whole homogenate is assayed, the differences did not always approach statistical significance. In 7 separate experiments (42 experimental and 42 control animals), we have been unable to demonstrate such an increase. We have repeated his work as closely as possible and have not been able to reproduce the results. The difference might be due to a difference in rat strain or to a subtle difference in methodology. Our failure to detect an increase in choline acetyltransferase in response to reserpine injection does not alter our main thesis that tyrosine hydroxylase is a more sensitive indicator of increased sympathetic nerve activity.

This work was supported by Grants N S-I 1310, NS-12121 and Program Project Grant HL-014388 from the U.S. Public Health Service.

1 Chuang, D. M. and Costa, E., Biosynthesis of tyrosine hydroxylase in rat adrenal medulla after exposure to cold, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 4570-4574. 2 Coyle, J. T., Tyrosine hydroxylase in rat brain: cofactor requirements, regional and subcellular distribution, Biochem. Pharmacol., 21 (1972) 1935-1944. 3 Dairman, W. and Udenfriend, S., Increased conversion of tyrosine to catecholarnines in intact rat following elevation of tissue tyrosine hydroxylase levels by administered phenoxybenzamine, Molec. Pharmacol., 6 (1971) 350-356. 4 de Groot, J., The Rat Forebrain in Stereotaxic Coordinates, Proc. Royal Dutch Academy o! Sciences, Dept. of Physics, 2nd Series, Part 52, no. 4, North-Holland Publishing, Amsterdam, 1959. 5 Folkow, B. and Rubenstein, E. H., Cardiovascular effects of acute and chronic stimulations of the hypothalamic defense area in the rat, Acta physiol, scand., 68 (1966) 48-57. 6 Fonnum, F., Choline acetyltransferase binding to and release from membranes, Biochem. J., 109 (1968) 389-398. 7 Hoeldtke, R. and Kaufman, S., Bovine adrenal tyrosine hydroxylase, J. biol. Chem., 252 (1977) 3160-3169. 8 Kvetfiansk3~, R., Weise, V. K. and Kopin, 1. J. , Elevation of adrenal tyrosine hydroxylase and phenylethanolamine-N-methyl transferase by repeated immobilization of rats, Endocrinology, 87 (1970) 744-749. 9 Lowry, O. H., Rosebrough, N. H., Farr, A. L. and Randall, R. J., Protein measurement with Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 197

l0 Mandell, A. J. and Morgan, M., Amphetamine induced increase in tyrosine hydroxylase activity, Nature (Lond.), 227 (1970) 75 76. 11 Molinoff, P. B. and Axelrod, J., Biochemistry of catecholamines, Ann. Rev. Biochem., 40 (1971) 465-50O. 12 Molinoff, P. B., Brimijoin, S., Weinshilboum, R. and Axelrod, J., Neurally mediated increase in dopamine-/3-hydroxylase activity, Proc. nat. Acad. Sci. (Wash.), 65 (1970)453458. 13 Morgenroth, V. H., Hegstrand, L. R., Roth, R. H. and Greengard, P., Evidence for involvement of protein kinase in the activation by adenosine-3':5'-monophosphate of brain tyrosine 3-mono- oxygenase, J. biol. Chem., 250 (1975) 1946 1948. 14 Mueller, R. A., Thoenen, H. and Axelrod, J., Adrenal tyrosine hydroxylase: compensatory increase in activity after chemical sympathectomy, Science, 163 (1969) 468469. 15 Mueller, R. A., Thoenen, H. and Axelrod, J., Increase in tyrosine hydroxylase activity after reserpine administration, ,1. Pharmacol. exp. Ther., 169 (1969) 74-79. 16 Mueller, R. A., Thoenen, H. and Axelrod, J., Inhibition of transsynaptically increased tyrosine hydroxylase activity by cycloheximide and actinomycin D, Molec. Pharmaeol., 5 (1969) 463-469. 17 Oesch, F., Trans-synaptic induction of choline acetyltransferase in the preganglionic neuron of the peripheral sympathetic nervous system, J. Pharmacol. exp. Ther., 188 (1974) 439-446. 18 Reis, D. J., Moorehead, D. T., Rifkin, M., Joh, T. H. and Goldstein, M., Changes in adrenal enzymes synthesizing catecholamines in attack behaviour evoked by hypothalamic stimulation in the cat, Nature (Lond.), 229 (1971) 562 563. 19 Roskoski, R., Jr., Choline acetyltransferase: evidence for an acetylenzyme intermediate, Bio- mistry, 12 (1973) 3709 3714. 20 Roskoski, R., Jr., Schmid, P. G., Mayer, H. E. and Abboud, F. M., In vitro acetylcholine biosynthesis in normal and failing guinea pig hearts, Circulut. Res., 36 (1975) 547-552. 21 Roskoski, R., Jr., Lund, D. D., Knuepfer, M. M., Brody, M. J., Schmid, P. G. and Bhatnagar, R. K., Tyrosine hydroxylase activity is a more sensitive indicator of sympathetic nervous system activation than choline acyltransferase activity, Fed. Proc., 37 (1978) 524. 22 Steel, R. G. D. and Torri, J. H., Principles and Procedures of Statistics, McGraw-Hill, New York, 1960, pp. 99-131. 23 Thoenen, H., Induction of tyrosine hydroxylase in peripheral and central adrenergic neurons by cold exposure of rats, Nature (Lond.), 228 (1970) 861 862. 24 Thoenen, H., Otten, U. and Oesch, F., Transsynaptic regulation of tyrosine hydroxylase. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, Oxford, 1973, pp. 179-185. 25 Weiner, N. and Mosimann, W. F., The effect of insulin on the catecholamine content and tyrosine hydroxylase activity of cat adrenal glands, Biochem. Pharmacol., 19 (1970) 1189-1199. 26 Weiner, N., Lee, F-L., Barnes, E. and Dreyer, E., Enzymology of tyrosine hydroxylase and the role of cyclic nucleotides in its regulation. In E. Usdin, N. Weiner and M. B. H. Youdim (Eds.), Structure and Function of Monoamine Enzymes, Marcel Dekker, New York, 1977, pp. 109-148.