In situ activity and phosphorylation of tyrosine hydroxylase in the median eminence

Molecular und Cellulur Endocrinoloa, Elsevier Scientific Publishers Ireland,

46 (1986) 21-27 Ltd.

MCE 01479

In situ activity and phosphorylation of tyrosine hydroxylase in the median eminence * John C. Porter Cecil H. and Idu Green Center for Reproductive Biology. Department of Obstetrics und Gynecolou, The University of Texas Health Science Center (II Dallas, Southw,estern Medical School, 5323 Hurry Hmes Bouleuurd, Dallas, TX 7523.5 (U.S.A.)

Key words:

DOPA

synthesis;

enzyme

(Received

14 January

activation;

veratridine

1986; accepted

20 February

1986)

Summary Intracellular activation and phosphorylation of tyrosine hydroxylase (TH; E.C. 1.14.16.2) in the median eminence of the rat brain were investigated. The in situ activity of TH was assayed by the accumulation of t-dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic fragments incubated in the presence of NSD 1015. When hypothalamic fragments were incubated with veratridine (O-1O-3 M), maximal stimulation of TH activity was observed at 10m4 M. The mean concentration of DOPA in the median eminence of hypothalamic fragments incubated with 10e4 M veratridine was 3 times that seen in its absence. Phosphorylation of TH in the median eminence was evaluated by autoradiographic quantification of [“PITH in 32P-labelled median eminence tissue. The amount of [“PITH in 32P-labelled median eminence incubated with 10e4 M veratridine was 2 times that seen in the absence of veratridine. These data are consistent with the view that in the median eminence phosphorylation and activation of TH are linked events and that phosphorylation may be a means of regulating the biosynthesis of dopamine in this region of the brain.

Introduction The secretion of dopamine by tuberoinfundibular neurons is believed to be limited by the availability of L-dihydroxyphenylalanine (DOPA). In the presence of exogenous DOPA, the secretion of dopamine by these neurons is 50 times that seen in its absence (Reymond and Porter, 1981). Conversely, inhibition of the transformation of DOPA to dopamine inhibits the release of dopamine (Reymond and Porter, 1982). Inhibition of the * This work was supported AG00306, and AGO4344 Health. Bethesda. MD. 0303-7207/86/$03.50

by Research Grant AM01237, from the National Institutes of

0 1986 Elsevier Scientific

Publishers

Ireland,

activity of tyrosine hydroxylase (TH) (tyrosine 3-monooxygenase, t-tyrosine,tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating, E.C. 1.14.16.2)), the enzyme that catalyzes the formation of DOPA from t_-tyrosine (Levitt et al., 1965). prevents the release of dopamine into portal blood (Gudelsky and Porter, 1979). These findings support the view that the rate of formation of DOPA from r_-tyrosine regulates the biosynthesis of dopamine. If so, it follows that the mechanism(s) that regulates the activity of TH is important in the control of dopamine secretion by tuberoinfundibular neurons. Through the use of a variety of paradigms, it has been found that the activity of TH is increased Ltd.

22

by phosphorylation (Joh et al., 1978; Yamauchi and Fujisawa, 1979; Edelman et al., 1981; Vigny and Henry, 1982; Cahill and Perlman, 1984). In the present study, we have investigated the hypothesis that the in situ activity and phosphorylation of TH in the median eminence are increased by a common stimulant and may be linked events. Materials and methods Animals Adult female rats (Long-Evans strain) ovariectomized at least 3 weeks before use, were housed in a temperature-controlled room (20-22°C) with a 14:lO h hght:dark schedule. Food and water were available at all times. Each rat was killed by decapitation, and the ventromedial hypothalamus or median eminence was dissected according to the procedure of Arita and Kimura (1984). Meusurement of TH activit), In situ activity of TH was assayed by the accumulation of DOPA in the median eminence (Arita and Kimura, 1984) using a 30-min pre-incubation before beginning the assay. The assay was conducted in the presence of 3-hydroxybenzylhydrazine (NSD 1015), an inhibitor of the activity of aromatic L-amino acid decarboxylase (Carlsson et al., 1972; Carlsson and Lindqvist, 1973). Each hypothalamic fragment was incubated in 1 ml of medium. The basic medium consisted of the following: 116 mM sodium chloride, 5.3 mM potassium chloride, 1.8 mM calcium chloride, 5 mM glucose, 0.8 mM magnesium sulfate, 10 PM monosodium phosphate, and 25 mM Hepes buffer, pH 7.4. The experimental medium was identical to the basic medium except for the presence of 10e5, M veratridine. The basic and 10-4, or lo-’ experimental media contained 10 mM NSD 1015 and 0.2 mM L-tyrosine. The incubation were conducted at 37°C under oxygen in an oscillating water bath. At the end of the incubation, the vials were chilled to 0°C and the median eminence was excised with the aid of a dissecting microscope. The median eminence was homogenized in 0.3 M perchloric acid containing 3 mM EDTA and 5 mM sodium metabisulfite. The homogenate was centrifuged for 1 min, and the supernate and

pellet were catecholamines centrifugations

used in the and protein, were conducted

quantification of respectively. (All at 10000 X 8.)

Measurement of DOPA, dopamine, norepinephrine, und protein DOPA, dopamine, norepinephrine, and epinephrine were quantified by means of high-pressure liquid chromatography with electrochemical detection (Bioanalytical Systems, West Lafayette, IN) using an adaptation of the method of Felice et al. (1978). Median eminence tissue containing approximately 25 pg protein was homogenized in 50 ~1 of 0.1 M perchloric acid (PCA) and centrifuged. Aliquots of the supernatant fluid and of the standard solution containing DOPA, dopamine, norepinephrine, or epinephrine in 0.1 M PCA were chromatographed without further processing on a Biophase ODS 5 pm column using a precolumn of 10 pm ODS-Hypersil (Upchurch Scientific, Oak Harbor, WA). The mobile phase consisted of 60 mM citric acid, 50 mM disodium phosphate, pH 3.0, 0.86 mM sodium octyl sulfate, 0.54 mM EDTA, and 6% methanol. (The composition of the mobile phase sometimes requires an adjustment of the concentration of methanol and/or sodium octyl sulfate to achieve optimal resolution on a particular column.) A flow of 1.2 ml/min of the mobile phase was employed. In a typical separation, retention times for norepinephrine, DOPA, epinephrine, and dopamine were 5.36, 6.36, 7.90, and 15.68 min, respectively. Radiolabelling the median eminence The median eminence was radiolabelled with 32P in a manner similar to that employed by Cahill and Perlman (1984) for superior cervical ganglia. Median eminence tissue was incubated in basic medium, defined earlier, containing ‘*P orthophosphoric acid (450 pCi/ml) at 37°C under oxygen in an oscillating water bath. After 2 h, the tissue was rinsed, placed in fresh medium or medium containing 10e4 M veratridine, and incubated at 37°C under oxygen. At the end of the incubation, each median eminence was homogenized in 1 ml of phosphatase-inhibiting and solubilizing solution (Cahill and Perlman, 1984) consisting of 30 mM potassium phosphate buffer, pH 7.5, 5 mM sodium fluoride, 1 mM EDTA. 0.5

23

mM phenylmethylsulfonyl Nonidet P40.

fluoride,

and

0.5%

Treatment of S. aureus S. uureus cells (Behring, Diagnostics, LaJolla, CA) were washed in 20 mM Tris buffer, pH 8.0, containing 100 mM sodium chloride, 1 mM EDTA, and 0.5% Nonidet P40 (Solution A). A 10% suspension of washed cells was prepared in Solution B (Solution A containing 1 mg BSA/ml). The function of the S. aureus cells was to adsorb the immunoglobulin in S828 antiserum, including the antibodies bound to TH. Immunoprecipitation of [“PITH Immunoprecipitation was conducted in the manner described by Jones (1980) modified as follows. An aliquot (940 ~1) of the homogenate of “P-1abelled median eminence tissue, known to contain 20-40 ng TH (Porter, 1986) was mixed with 1 ~1 of pre-immune rabbit serum or rabbit S828 antiserum. (One ~1 of S828 antiserum has the capacity to bind 135 ng TH (Porter, 1986).) The solution was incubated overnight at 4°C before mixing with 30 ~1 of the S. aweus suspension. The mixture was incubated at 0°C for 30 min and centrifuged. The pelleted cells were washed and then suspended in Solution C (Solution A containing 0.1% sodium dodecylsulfate (SDS)). The suspension was layered on 10 volumes of Solution D (Solution C containing 1.5 M sucrose) and centrifuged. The pelleted cells were washed 3 times with Solution C, and once with Solution E (Solution A diluted 9 times with water). The washed cells were suspended in 30 ~1 of Laemmli (1970) sample buffer, heated in sealed tubes for 2 min in a boiling water bath, and centrifuged. The supernatant fluid was subjected to electrophoresis. Prepuration of [“PITH marker from purified TH TH was purified according to the procedure of Okuno and Fujisawa (1982) from tumor tissue grown in rats (New England Deaconess Hospital strain, Boston, MA) inoculated subcutaneously with PC-12 cells (Greene and Tischler, 1976). Purified TH was phosphorylated in a manner similar to that used by Yamauchi and Fujisawa (1979). The reaction mixture consisted of 200 ~1 of 20 mM sodium phosphate buffer, pH 6.8, containing

2 pg TH, 2 PCi [Y-~~P]ATP (spec. act. 3000 Ci/mmol), 5 PM CAMP, 5 mM magnesium chloride and 5 pg CAMP-dependent protein kinase from beef heart. After a 5-min incubation at 30°C S828 antiserum (40 ~1) was added to the reaction mixture and allowed to stand overnight at 4°C. Subsequent events used in the purification of [“PITH were identical to those employed when using 32P-labelled median eminence tissue except that the amounts of all reagents were increased to accommodate the quantity of TH that was phosphorylated. Electrophoresis Electrophoresis was performed on polyacrylamide gel slabs (1.5 mm thick, 7.5% gel) in the presence of 0.1% SDS. The stacking gel was formed using a 20-well comb where each well was 3.35 mm wide. The sample volume ranged from 10 ~1 to 30 ~1. The gels were calibrated with the following molecular weight standards (BioRad, Richmond, CA): phosphorylase b, 92 500; BSA, 66 200; ovalbumin, 45 000; carbonic anhydrase, 31000; soybean trypsin inhibitor, 21500; and lysozyme, 14400. Rudiouutogruphy and analysis After electrophoresis, the slab was fixed and stained for 30 min in 25% methanol containing 10% acetic acid and 0.05% Coomassie blue. After destaining, the slab was immersed for 30 min in 10% acetic acid containing 1% glycerol and dried by means of a slab dryer (BioRad). The dry slab to Kodak XAR-2 film was exposed at -70°C using an intensifying screen (DuPont Cronex Xtra Life). The developed film was scanned by means of a densitometer (Helena Laboratories, Beaumont, TX). The data were analyzed statistically by Student’s t-test (Fisher, 1954). Results TH activity The time-dependent accumulation of DOPA in the median eminence of hypothalamic fragments incubated in the presence of NSD 1015 is illustrated in Fig. 1. Between 0 and 60 min, the concentration of DOPA increased linearly. Therefore, a 60-min incubation was used in the assay of

I-

200

1 5

2

e P

0

15

45

30

norepinephrine was low at 0 time and lower still at 60 min (Fig. 1). Epinephrine was not detected. Incubation or hypothalamic fragments in the presence of 0, lop5 M, or 1O-4 M veratridine resulted in a dose-dependent increase in the amount of DOPA in the median eminence (Fig. 2). In the presence of 10e3 M veratridine, the amount of DOPA in the median eminence was less than that seen with lop4 M veratridine.

A

60

B

C

D

E

Minutes

Fig. 1. In vitro accumulation of DOPA in the median eminence of hypothalamic fragments as a function of time. The incubations were conducted in the presence of 10 mM NSD 1015. Closed circles represent means (n = 3); vertical lines denote the magnitude of the standard errors.

the in situ activity of TH. The amount of dopamine in the median eminence decreased throughout the incubation period; the amount of

kDa g2-

‘;$

66_

.i:

_’‘,

i’

TH - $

(91

45-

T

iII

31 -

(9)

19)

(9)

-

I -

0

-

10-5

Veratridine

-

10-4

-

__I

to-3

(M)

Fig. 2. In vitro accumulation of DOPA in the median eminence of hypothalamic fragments as a function of the concentration of veratridine in the incubation medium. The incubation was conducted for 60 min in the presence of 10 mM NSD 1015. Height of the bars represents means; the vertical lines denote standard errors. The number of hypothalami used is indicated in parentheses.

Fig. 3. Evaluation of the specificity of S828 antiserum for radiolabelled TH in homogenates of ‘2P-labelled median eminence tissue. lmmunoprecipitates of homogenates of radiolabelled median eminence tissue were electrophoresed on polyacrylamide gel slabs under denaturing conditions. The film was exposed to the slab for 7 days at -70 C. Autoradiograms of immunoprecipitates prepared under different conditions are illustrated in the following columns: column A. 1 ~1 pre-immune serum; column B, 1 ~1 of S828 antiserum; column C, 1 ~1 of S828 antiserum and 500 ng purified TH; column D. 1 ~1 of S828 antiserum and 5000 ng purified TH. The location of purified TH electrophoresed simultaneously with the immunoprecipitates is indicated in column E. The locations of the molecular weight markers are indicated on the left. Purified TH and the molecular weight markers were identified by staining with Coomassie blue.

Phosphovylation of TH To test the specificity of the antiserum for [ 32PITH, 32P-labelled median eminence tissue from 12 rats was homogenized in phosphatase-inhibiting buffer, and the supernate was divided into 12 aliquots. Immunoprecipitation of [ 32P]TH in an aliquot was conducted in the presence of 1 ~1 of pre-immune rabbit serum or 1 ~1 of S828 antiserum containing 0, 500, or 5000 ng of purified TH. An autoradiogram of a gel slab on which these immunoprecipitates were electrophoresed is presented in Fig. 3. In samples treated with preimmune rabbit serum, no radiolabelled substance was recovered; whereas in samples treated with S828 antiserum, one radiolabelled substance was seen. This radiolabelled substance co-migrated with purified TH. When the immunoprecipitation with S828 antiserum was conducted in the presence of 500 ng or 5000 ng of purified TH, the intensity of the band attributed to [32P]TH was greatly reduced compared to that seen in the absence of exogenous TH. In another experiment, an autoradiogram of immunoprecipitates of 32Plabelled median eminence tissue revealed the presence of one major radiolabelled substance which co-migrated with authentic [32P]TH (data not shown). To optimize the time of phosphorylation, median eminence tissue was incubated in the pres-

l20

t

.-.-. / :

I:: 0

4

8

12

i6

a0

CONTROL

m

VERATRIDINE,10-4

M

Fig. 5. Effect of veratridine on the phosphorylation of TH in ‘*P-labelled median eminence tissue. The film was exposed to the slab for 4 days, and the extent of the incorporation of 32P into TH is expressed in arbitrary units. The height of the bars denotes means: the vertical lines represent the magnitude of the standard errors. The number of median eminences is shown in parentheses.

ence of lop4 M veratridine for 2, 5, 10, or 20 min and analyzed for [ 32P]TH (Fig. 4). The amounts of [“PITH formed after 5 and 10, and 20 min were essentially the same; therefore, the duration of the phosphorylation was standardized at 10 min. To determine whether conditions that stimulate the in situ activity of TH also stimulate phosphorylation of TH, 18 median eminences were co-incubated with 32P for 2 h. Then, 9 of these 18 median eminences were incubated in basic medium containing lop4 veratridine, and 9 were incubated in basic medium only. After 10 min, all were simultaneously processed for [ 32P]TH to insure equal time of exposure of all samples to the film. As shown in Fig. 5, incubation of median eminence tissue with lop4 M veratridine resulted in a significant (P < 0.001) increase in the phosphorylation of TH in the median eminence compared to the control.

20

MINUTES Fig. 4. Incorporation of 32P into TH by radiolabelled median eminence tissue as a function of time. The incubation was conducted in the presence of 10m4 M veratridine. 32P incorporation into TH is expressed in arbitrary units. Each symbol denotes the average of results from 2 median eminences.

Discussion

The evidence that TH is phosphorylated in the median eminence is as follows. First, through use of specific antiserum against TH, polyacrylamide gel electrophoresis, and radioautography, im-

munoprecipitates of homogenates of 32P-labelled median eminence tissue were found to contain one radiolabelled substance. This substance had a molecular weight of about 60000, the approximate molecular weight of monomeric TH. Second, this radiolabelled substance and purified TH competed for binding sites on antibodies against TH. Third, this radiolabelled substance and authentic [“PITH co-migrate when subjected to polyacrylamide gel electrophoresis. Thus, it is concluded that TH is phosphorylated in median eminence tissue. Using rat superior cervical ganglia, Cahill and Perlman (1984) and Cahill et al. (1985) found that several conditions, including stimulation with veratridine, result in increased intracellular activity and phosphorylation of TH. In the present investigation, it was found that incubation of median eminence tissue with veratridine (10m4 M) caused a 3-fold increase in the enzymic activity and a 2-fold increase in the phosphorylatioll of TH. Although these data do not provide indisputable evidence that the increased intracellular activity of TH induced by veratridine was a consequence of phosphorylation of TH, the evidence is consistent with this view. Support for this interpretation is provided by others who found that phosphorylation of purified TH potentiates its activity (Edelman et al., 1978. 1981; Joh et al., 1978; Yamauchi and Fujisawa, 1979; Markey et al., 1980; Vulliet et al., 1980: Vigny and Henry, 1982). In addition, conditions that lead to protein phosphorylation and presumably to phosphorylation of TH - result in an increase in TH activity (Harris et al., 1974; Lovenberg et al., 1975; Morgenroth et al., 1975; Ames et al., 1978). The mechanism of the activation of TH by phosphoryiation has been variously interpreted. Some investigators have found that phosphorylation of TH results in a reduction of the apparent K,,, of the enzyme for the pterin co-factor (Edelman et al., 1978; Markey et al., 1980; Vulliet et al., 1980); others have found an increase in the k’,,, of the enzyme with little change in the K, (Joh et al., 1978). The effect of phosphorylation on TH activity may be due to a change in the pH optimum of the enzyme (Pollock et al., 1981). Vigny and Henry (1982) have presented an

interesting hypothesis to explain the effect of phosphorylation of TH on its activity. They propose that a cationic site is near the catalytic site of the enzyme. The positive charge of the cationic site impedes the interaction of the positively charged pterin co-factor with the catalytic site. thereby limiting the availability of the co-factor. They suggested that phosphorylation of TH reduces the effective charge of the cationic site. There is evidence that phosphorylation of TH frees the enzyme of end-product inhibition by dopamine as well as other catecholamines (Ames et al., 1978; Okuno and Fujisawa, 1985). The importance of this mechanism in the regulation of the activity of TN in the median eminence is not known. Although there is a relatively large quantity of TH in the median eminence of the rat (Porter, 19X6), it is not known whether the activities of all molecules of TH within a cell are equal. If phosphorylation is an important mechanism for regulating the activity of TH, it is likely that some TH molecules in a cell are phosphorylated and some are not. Therefore, it will be worthwhile to investigate the relationship between the extent of the phosphorylation of TH and the quantity of TH in the median eminence. Recently, it was found that the amount of TH in the median eminence of old female rats was greater than that in young female rats (Porter, 1986). This finding was unexpected since young female rats secrete more dopamine into portal blood than do old female rats (Reymond and Porter. 1981). In addition, the amount of TH in the median eminence of proestrous rats is greater than that of estrous rats (Porter, 1986). Yet, the secretion of dopamine into portal blood by estrous rats is greater than that by proestrous rats (BenJonathan et al., 1977). These findings present an incongruity between amount and activity of intracellular TH. It is interesting to speculate that the activity of TH may be modulated by a mechanism involving phosphorylation, thereby regulating the rate of secretion of dopamine. Acknowledgements

The Moore,

author Nhu-Y

thanks Dong,

Sharyn Monroe, Mary S. and Robert Lipsey for su-

27

perb technical assistance and Kay Stanley for excellent editorial assistance. Veratridine used in this study was a gift from Mr. Ralph Salkin (Pennick Corporation, Lyndhurst, NJ) and from Dr. Mathew Suffness (Natural Products Branch, Division of Cancer Treatment, NCI, Bethesda, MD). The author is appreciative of their generosity. PC-12 cells were provided by Dr. Lloyd A. Greene, Department of Pharmacology, New York University School of Medicine. References Ames, M.M., Lerner, P. and Lovenberg, W. (1978) J. Biol. Chem. 253, 27-31. Arita, J. and Kimura, F. (1984) Neuroendocrinology 39, 524-529. Ben-Jonathan. N., Oliver. C., Weiner, H.J., Mical, R.S. and Porter, J.C. (1977) Endocrinology 100, 4522458. Cahill, A.L. and Perlman. R.L. (1984) Biochim. Biophys. Acta 805, 217-226. Cahill, A.L., Horwitz, J. and Perlman, R.L. (1985) J. Neurothem. 44, 680-685. Carlsson. A. and Lindqvist, M. (1973) J. Neural Transm. 34. 79-91. Carlsson. A., Davis, J.N., Kehr. W., Lindqvist, M. and Atack, C.V. (1972) Naunyn-Schmied. Arch. Pharmacol. 275. 153-168. Edelman, A.M., Raese. J.D., Lazar, M.A. and Barchas. J.D. (1978) Commun. Psychopharmacol. 2, 462-465. Edelman. A.M., Raese, J.D., Lazar, M.A. and Barchas, J.D. (1981) J. Pharmacol. Exp. Ther. 216, 647-653. Felice, L.J., Felice, J.D. and Kissinger, P.T. (1978) J. Neurothem. 31, 1461-1465. Fisher, R.A. (1954) In: Statistical Methods for Research Workers, 12th edn, Ed.: F.A.E. Cred (Hafner Publishing Co., New York) pp. 114-173.

Greene, L.A. and Tischler. A.S. (1976) Proc. Natl. Acad. Sci. U.S.A. 13, 2424-2428. Gudelsky, G.A. and Porter, J.C. (1979) Endocrinology 104. 583-587. Harris, J.E.. Morgenroth, III, V.M.. Roth, R.N. and Baldessarini. R.J. (1974) Nature 252. 156-158. Joh. T.H., Park, D.H. and Reis, D.J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4744-4748. Jones, P.P. (1980) In: Selected Methods in Cellular Immunology. Eds.: B.B. Mishell and S.M. Shiigi (W.H. Freeman and Company) pp. 398440. Laemmli, U.K. (1970) Nature 227. 680-685. Levitt. L.. Spector, S., Sjoerdsma, A. and Udenfriend, S. (1965) J. Pharmacol. Exp. Ther. 148, l-8. Lovenberg, W.. Bruckwick. E.A. and Hanbauer, I. (1975) Proc. Natl. Acad. Sci. U.S.A. 72. 295552958. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Markey, K.A.. Kondo, S., Shenkman, L. and Goldstein, M. (1980) Mol. Pharmacol. 17, 79985. Morgenroth. III. V.H.. Hegstrand. L.R.. Roth. R.H. and Greengard. P. (1975) J. Biol. Chem. 250. 194661948. Okuno. S. and Fujisawa. H. (1982) Eur. J. Biochem. 122, 49-55. Okuno, S. and Fujisawa, H. (1985) J. Biol. Chem. 260, 2633-2635. Pollock, R.J., Kapatos. G. and Kaufman. S. (1981) J. Neurothem. 37, 855-860. Porter, J.C. (1986) Endocrinology (in press). Reymond. M.J. and Porter, J.C. (1981) Brain Res. Bull. 7. 69-73. Reymond, M.J. and Porter, J.C. (1982) Endocrinology 111. 1051-1056. Vigny, A. and Henry. J.P. (1982) Biochem. Biophys. Res. Commun. 106, l-7. Vulliet, P.R., Langan, T.A. and Weiner, N. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 92-96. Yamauchi, T. and Fujisawa. H. (1979) J. Biol. Chem. 254. 640886413.