Journal of Pineal Research 6:285-292 (1989)

Measurement of Tryptophan Hydroxylase Activity in Rat Pineal Glands and

Pinealocytes Using an HPLC Assay With Electrochemical Detection

David Sugden, Richard Grady, Jr., and Ivan N. Mefford Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National

Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (D.S.) and Department of Chemistry, Boston College, Chestnut

Hill, Boston, Massachusetts (R.G., I.N.M.)

A method for measuring tryptophan hydroxylase activity by assaying the product 5-HTP using high-performance liquid chromatography (HPLC) with electrochemical detection is described. A nocturnal elevation (80% ) in rat pineal gland tryptophan hydroxylase activity was detected. Experiments on isolated rat pinealocytes with the protein synthesis inhibitor cycloheximide indicate that tryptophan hydroxylase turns over rapidly in these cells. This method will be valuable in studies of the adrenergic mechanisms regulating pineal tryptophan hydroxylase activity.

Key words: diurnal rhythm, adrenergic regulation, cycloheximide

INTRODUCTION

The regulation of tryptophan hydroxylase activity in the rat pineal gland has been studied by a number of investigators. Initial studies indicated that enzyme activity did not show diurnal changes [Deguchi, 19771 or increase in activity following adrenergic stimulation [Bensinger et al., 1974; Chan and Ebadi, 19811; more recent studies have reported a circadian rhythm in activity, with enzyme activity increasing during the dark period [Shibuya et al., 19781 and adrenergic stimulation of activity in vitro [Sitaram and Lees, 1978, 19841. These studies have indicated that noradrenaline (NA), acting through a p- adrenergic receptor, may be the transmitter causing this increase in activity

Received June 21, 1988; accepted September 2, 1988.

Address reprint requests to Dr. David Sugden at his present address, Department of Physiology, King’s College, London W 8 7AH, England.

Ivan N. Mefford is presently at the National Institutes of Mental Health, Bethesda, MD 20892.

Richard Grady Jr. is presently at the Upjohn Co., Kalamazoo, MI 49009

0 1989 Alan R. Liss, Inc. *

286 Sugden et al.

[Shibuya et al., 1978; Sitaram and Lees, 19841, but little data is available on the intracellular mechanisms involved. The present study describes a method for measuring tryptophan hydroxylase by assay of 5-hydroxytryptophan (5-HTP) by high-performance liquid chromatography (HPLC) with electrochemical detection and illustrates the utility of this assay method for studies on the regulation of tryptophan hydroxylase in rat pineal glands and pinealocytes in suspension culture.

MATERIALS AND METHODS Materials

Cycloheximide, catalase (from bovine liver, 1 X lo6 U/ml), L-tryptophan, L-5-hydroxytryptophan, and DL-6-methyl-5,6,7,8-tetrahydropterine were ob- tained from Sigma. Other reagents were purchased from sources previously identified [Vanecek et al., 19851.

Preparation of Rat Pinealocytes

Female Sprague-Dawley rats ( 50-1 00 animaldbatch, approximately 7 weeks old, 200-250 gm) were purchased from Charles River Breeding Labora- tories (Wilmington, MA). Rats were reared under diurnal lighting conditions and placed in a 12:12 L:D environment (lights on 6 am) on arrival for 7 days before use. Pinealocytes were prepared by trypsinization as previously de- scribed [Buda and Klein, 1978; Vanecek et al., 19851, with minor modifications. Glands were incubated with trypsin (1 mg/ml, type 111, Sigma) for 15 min, and then titurated and reincubated for a further 10 min. Ascorbate (0.1 mg/ml) and DL-propranolol (1 pM) were included in the trypsinizing medium. Cells were washed twice following dispersion to remove propranolol from the medium.

Cells were incubated (8 X 104/ml, 37"C, 95% air/5% COz) in suspension culture in Dulbecco's modified Eagle's medium (DMEM) containing fetal calf serum (10% v/v). Viability judged by trypan blue exclusion on the first day of culture exceeded 95% in all experiments. Aliquots of cells (0.5 ml, 4 x lo4 cells) were treated as indicated in the figure legends and then collected by centrifugation (S,OOOg, 30 sec) and frozen ( - SOOC) until assayed for trypto- phan hydroxylase.

Tryptophan Hydroxylase Assay

Tryptophan hydroxylase was assayed by quantitating the amount of 5-HTP formed from L-tryptophan by HPLC with electrochemical detection [ Mefford and Barchas, 19801. Pinealocytes or individual pineal glands were disrupted by brief sonication (< 10 sec, 4°C) in 120 pl of sodium phosphate buffer (50 mM, pH 6.8) and the homogenate immediately used as the enzyme source. Routinely the assay mixture (80 pl) containing enzyme (5-40 pl), L-tryptophan (0.4 mM), DL-6-methyl-5,6,7,8-tetrahydropterine (0.5 mM), dithiothreitol (2 mM), catalase (1,600 U), and NSD 1015 ( 1 mM) was incubated at 37°C for 30 min. The reaction was stopped by the addition of ice-cold perchloric acid (20 pl, lN), and the precipitate was removed by centrifugation (l2,000g, 2 min). All samples were assayed in duplicate. An aliquot of clear supernatant

HPLC Assay of Pineal Tryptophan Hydroxylase 287

was loaded onto an Ultrasphere ODS 7.5 X 0.46 cm, 3-bm column (Altex/ Beckman, Berkely, CA). Isocratic elution was accomplished during 0.1 M ammonium acetate, 0.1 M acetic acid, and 2% acetone (v/v). The flow rate ( 1 mumin) was maintained using a Milton Roy minipump with pulse damping. The detector was an amperometric controller (LC-2A, Bioanalytical Systems Inc., West Lafayette, IN), and the separated product 5-HTP was oxidized at a glassy carbon electrode at a potential of + 0.5 V versus an Ag/AgCl reference electrode. The identity of 5-HTP, eluting at 3.2 min, was confirmed by matching retention times with authentic standard. Calibration curves established that the detector response was linear between 0.01 and 100ng of standard injected. Enzyme activities were determined in duplicate.

NAT activity was measured in pineal homogenates using a radioenzymatic assay [Sugden et al., 19831, and protein was determined using a dye-binding method [Bradford, 19761.

RESULTS Tryptophan Hydroxylase Assay

The oxidation of hydroxyindoles such as 5-HTP at a carbon electrode surface can be accomplished at + 0.5 V (vs. Ag/AgCI reference electrode), while a potential of + 0 . 9 V is required to oxidize the tryptophan hydroxylase substrate L-tryptophan (Fig. 1 ). Thus, irrespective of their different elution times, a potential of 0.5 V selectively detects the enzyme product 5-HTP and not the substrate, tryptophan. Inclusion of NSD 101 5, an inhibitor of aromatic amino acid decarboxylase, in the enzyme assay prevented the further degradation of the product 5-HTP to 5-HT. This finding was confirmed by measurement of 5-HT (retention time 4.2 min, detector potential +0.5V) by HPLC with electrochemical detection. N o increase in 5-HT was seen when the enzyme was incubated with the reaction mixture at 37°C for up to 30 min (data not shown). None of the other components of the enzyme assay (NSD 101 5 or DL-6-methyl- 5,6,7,8-tetrahydropterine) interferred with the electrochemical detection of 5-HTP. Sample chromatograms obtained with a 5-HTP standard and a trypto- phan hydroxylase assay sample are shown in Figure 1. The production of 5-HTP was shown to be linear with respect to time and the concentration of enzyme used (Fig. 2) . The 5-HTP content of the cell or tissue homogenate (i.e., endogenous 5-HTP content) was very low, as has been reported previously [Mefford and Barchas, 19801 and accounted for < 2% of the 5-HTP generated in the standard enzyme assay. Nevertheless, in all assays a sample of each enzyme was incubated with the reaction mixture at 4°C for 30min, and endogenous 5-HTP values were subtracted from the 5-HTP formed in the assay. The assay was very sensitive; activity could be detected using only 5 pg of the homogenate protein, corresponding to one-twentieth of a rat pineal gland. This high sensitivity permits the measurement of other pineal enzymes (NAT and hydroxyindole-0-methyltransferase) or products ( melatonin) in the same ho- mogenate.

Rat Pineal Tryptophan Hydroxylase A clear increase in pineal tryptophan hydroxylase activity was detected at

midnight compared with daytime (noon) values. As expected, the pineals used

288 Sugden et al.

-- 0 1 2 3 1 0 1 2 3 4 5 8

M I N U T E S M I N U T E S

Fig. 1. Chromatograms of 5-HTP standard (left) and tryptophan hydroxylase assay sample from pineal (right). 5-HTP (peak A: 4.26ng, left; 3.54ng, right) and 5-HT (peak B in pineal sample) were eluted and detected as described in Materials and Methods.

in these experiments also showed a marked (> 200-fold) increase in NAT activity at night (Table 1).

Tryptophan Hydroxylase Activity in Isolated Rat Pinealocytes

Tryptophan hydroxylase activity could be readily demonstrated in isolated rat pinealocytes maintained in a suspension culture. Enzyme activity was linearly related to cell density up to lo5 cells/sampIe and could be detected using as few as lo4 cells (Fig. 3). Assays of pineal tryptophan hydroxylase at different times after cell preparation showed that during the first 24 h of cell culture, enzyme activity increased two- to threefold and reached a plateau by 24h. A slight decline in activity by 48h, which was occasionally seen, is probably related to a decrease in cell viability at this time (approximately 90% ). Interestingly, the addition of the protein synthesis inhibitor cyclohexi- mide (30 pM) at 24 h, after enzyme activity had reached a maximum, reduced enzyme activity within 6 h (Fig. 4). Furthermore, cycloheximide (30 pM) prevented the initial rise in enzyme activity (data not shown).

HPLC Assay of Pineal Tryptophan Hydroxylase 289

1 . L

0 10 2 0 30 0 10 20 40

T I M E (min) ENZYME (PI)

Fig. 2. Linearity of tryptophan hydroxylase assay with time (a) and enzyme (b). Four pineal glands were removed from adult rats and sonicated in sodium phosphate buffer (50 mM, pH 6.8, 1 gland/200 pl). In a, aliquots of 40 p1 of this sonicate were assayed for the times indicated; in b, each of the enzyme samples were assayed for 30 min. Each point represents the mean of duplicate samples.

TABLE 1. Daymight Difference in Rat Pineal N-Acetyltransferase and Tryptophan Hy- droxylase Activities’

N-acet yltransferase Tryptophan hydroxylase (nmoVh/mg protein) (nmol/h/mg protein)

Day 0.2 2 0.1 20.0 2 1.6 Night 40.8 t 6.2* 36.3 t 1.9’

‘Male Sprague-Dawley rats (200-250 gm, Zivic-Miller Co., Allison Park, PA) had access to food and water ad libitum and were housed for 3 weeks under a diurnal lighting cycle (1ight:dark 14:10, lights on at 5 am). Rats were killed by decapitation at midnight and at noon, and the pineal glands were rapidly removed, frozen on dry ice, and stored at -80°C until they were assayed for tryptophan hydroxylase and N-acetyltransferase activities, as described in Materials and Methods. Values represent mean * SEM determined in eight rats at each time point. ‘“Night” values of both enzymes were significantly greater (P < 0.05) than “day” values, using Student’s t test.

DISCUSSION

Studies on the adrenergic regulation of the enzymes involved in melatonin synthesis in the rat pineal gland have focused primarily on the regulation of NAT (Klein et al., 1981; Sugden and Klein, 19861. Large changes in NAT activity in the rat pineal are of critical importance in generating the characteristic nocturnal rhythm in melatonin synthesis and secretion in this species. However, other species fail to show such large amplitude changes in NAT activity at night [Binkley, 19811, and it has been suggested that other mechanisms may contrib- ute to the regulation of the nocturnal elevation of melatonin in these species [Namboodiri et al., 1985a,b; Sugden et al., 19851. Indeed, other enzymes in the synthetic pathway are probably also adrenergically regulated. In vivo studies have indicated that HIOMT, the final step in melatonin synthesis, is regulated by the pineal P-adrenergic receptor [ Sugden and Klein, 19831. Nightly stimulation of the pineal by NA released from the sympathetic nerves that innervate the gland serves to maintain HIOMT activity at a high level. Studies of tryptophan hydroxylase regulation in the pineal have given conflicting results (see

290 Sugden et al.

0.4

t 0.3

e m > -

s- 0.2

: 0.1

S \

= z

4-

P > I- L

C

0 20 40 60 80

cell number ( x 1 0 3 )

Fig. 3. Linearity of pinealocyte tryptophan hydroxylase with cell density. Pinealocytes were prepared as described in Materials and Methods and aliquoted into microfuge tubes (1-8 X lo4 cells/0.5 ml). Cells were collected by centrifugation (8,OOOg, 30 sec), and the cell pellet was rapidly frozen on dry ice and stored at -80°C until assayed for tryptophan hydroxylase. Cell extracts were prepared by sonication (< 10 sec, 4°C) in sodium phosphate buffer (50 mM, pH 6.8). Each point represents the mean of three cell aliquots. Error bars fell within the area covered by the symbols.

0 12 24 36 48

TIME ( h )

Fig. 4. Changes in pinealocyte tryptophan hydroxylase activity after cell preparation: effect of cycloheximide. Pinealocytes (4 X lo4 cells) were treated as described in the legend to Figure 3. Cycloheximide (30 kM) was added at 24 h (arrow). Each point represents the mean k SEM of three cell aliquots assayed in duplicate.

Introduction). The present study supports the idea that tryptophan hydroxylase is adrenergically regulated in that an 80% increase in activity was apparent at midnight, compared with midday.

Tryptophan hydroxylase activity in pinealocytes was found to increase following cell preparation, reaching stable values after 24h. Why enzyme

HPLC Assay of Pineal Tryptophan Hydroxylase 291

xtivity should increase in this way is not known. Conceivably the increase in activity may represent a recovery of the enzyme from a detrimental effect of the trypsinization procedure used to isolate the cells. However, other pinealocyte proteins (NAT and HIOMT), including those located in the cell membrane (adenylate cyclase and p- and a1 -adrenoceptors), appear to be functionally intact within a few hours of cell preparation. The mechanism of the increase in activity appears to involve an increase in protein synthesis, since cyclohexi- mide, at a concentration known to block protein synthesis in these cells [Bensinger et al., 19741, completely prevented the rise in activity. Indeed, cycloheximide, when added to cells 24 h after their preparation, also dramati- cally reduced activity. These data suggest that tryptophan hydroxylase activity in pinealocytes, in contrast t o , the enzyme in brain tissue, turns over very rapidly, a suggestion that is in agreement with previous work using pineal explants in organ culture [Bensinger et al., 1974; Sitaram and Lees, 19781 and in vivo experiments [ Deguchi and Barchas, 19721.

The availability of the sensitive assay described in this report should allow the receptor and intracellular mechanisms regulating tryptophan hydroxylase in the rat pineal to be clarified by enabling studies on the adrenergic regulation of activity to be done in isolated pinealocytes. It has been speculated that the physiological importance of the rise in tryptophan hydroxylase activity at night in the rat pineal may be to compensate for the depletion of serotonin that occurs following the large increase in NAT activity [Sitaram and Lees, 19781. However, not all species show a large nighttime increase in NAT activity [Binkley, 19811; thus the possibility that a nocturnal elevation of tryptophan hydroxylase activity may contribute to the production of the circadian rhythm in pineal melatonin synthesis and secretion in these species must be considered.

LITERATURE CITED

Bensinger, R.E., D.C. Klein, J.L. Weller, W. Lovenberg ( 1974) Radiometric assay of total tryptophan hydroxylation by intact cultured pineal glands. J. Neurochem. 23:l 11-1 17.

Binkley, S. ( 1981 ) Pineal biochemistry: Comparative aspects and circadian rhythms. In: The Pineal Gland. I: Anatomy and Biochemistry. R.J. Reiter, ed. CRC Press, Boca Raton, FL, pp. 155-172.

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

Buda, M., D.C. Klein ( 1978) A suspension culture of pinealoqtes: Regulation of N-acetyltransferase activity. Endocrinology 103: 1483-1493.

Chan, A,, M. Ebadi (1981) Effects of norepinephrine on pineal tryptophan hydroxylase using an improved [ ''C]C02-trapping microtechnique. J. Pharmacol. Methods. 6:13-20.

Deguchi, T. (1977) Tryptophan hydroxylase in pineal gland of rat: Post-synaptic localization and absence of circadian change. J. Neurochem. 28:667-668.

Deguchi, T., J. Barchas (1972) Effect of p-chlorophenylalanine on tryptophan hydroxylase in rat pineal. Nature 23592-93.

Klein, D.C., D.A. Auerbach, M.A.A. Namboodiri, G.H.T. Wheler ( 1981) Indoleamine metabolism in the mammalian pineal gland. In: The Pineal Gland. I: Anatomy and Biochemistry. RJ. Reiter, ed. CRC Press, Boca Raton, FL, pp. 199-227.

Mefford, I.N., J.D. Barchas (1980) Determination of tryptophan and metabolites in rat brain and pineal tissue by reversed phase high performance liquid chromatography with electrochem- ical detection. J. Chromatogr. 181:187-193.

Namboodiri, M.A.A., D. Sugden, D.C. Klein, R. Grady Jr., I.N. Mefford (1985a) Rapid nocturnal increase in ovine pineal N-acet yltransferase activity and melatonin synthesis Effects of cycloheximide. J. Neurochem. 45:832-835.

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Namboodiri, M.A.A., D. Sugden, D.C. Klein, L. Tamarkin, LN. Mefford (198515) Serum melatonin and pineal indoleamine metabolism in a species with a small dayhight N-acetyltransferase rhythm. Comp. Biochem. Physiol. 80B:73 1-736.

Shibuya, H., M. Toru, S. Watanabe (1978) A circadian rhythm of tryptophan hydroxylase in rat pineals. Brain Res. 138364-368.

Sitaram, B.R., G.J. Lees (1978) Diurnal rhythm and turnover of tryptophan hydroxylase in the pineal gland of the rat. J. Neurochem. 31:1021-1026.

Sitaram, B.R., G.J. Lees (1984) Effect of oxygen on the induction of tryptophan hydroxylase by adrenergic agents in organ cultures of rat pineal gland. J. Neurochem. 42:1183-1185.

Sugden, D., D.C. Klein (1983) P-Adrenergic receptor control of rat pineal hydroxyindole-0. methyltransferase. Endocrinology 1 13348-353.

Sugden, D., D.C. Klein (1986) Adrenergic regulation of cyclic AMP and cyclic GMP in rat pinealocytes. in: Pineal and Retinal Relationships. P.J. O'Brien and D.C. Klein, eds. Academic Press, Orlando, FL, pp. 293-305.

Sugden, D., M.A.A. Namboodiri, J.L. Weller, D.C. Klein ( 1983) Melatonin synthesizing enzymes: Serotonin N-acetyltransferase and hydroxyindole-0-methyltransferase. In: "Methods in Biogenic Amine Research', S. Parvez, T. Nagatsu, I. Nagatsu, and H. Parvez, eds. Elsevied North Holland Biomedical Press, Amsterdam, pp. 567-572.

Sugden, D., M.A.A. Namboodiri, D.C. Klein, J.E. Pierce, R. Grady Jr., I.N. Mefford (1985) Ovine pineal a,-adrenoceptors: Characterization and evidence for a functional role in the regula- tion of serum melatonin. Endocrinology 116:1960-1967.

Vanecek, J., D. Sugden, J.L. Weller, D.C. Klein (1985) Atypical synergistic P- and al-adrenergic regulation of adenosine 3,5'-cyclic monophosphate and guanosine 3',5'-cyclic monophos. phate in rat pinealocytes. Endocrinology 116:2167-2173.