Protein synthesis in pineal gland during serotonin-N-acetyltransferase induction

OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 1, November, pp. 1-7, 1978

ARCHIVES

Protein

Synthesis

JEREMIAH Section on Biochemical

in Pineal Gland during Induction J. MORRISSEY’ Pharmacology,

Serotonin-N-Acetyltransferase

and WALTER

LOVENBERG

National Heart, Lung, and Blood Institute, Maryland 20014

Received February

NIH, Bethesda,

24, 1978; revised May 15, 1978

Protein synthesis in the cultured rat pineal gland was monitored during the course of Nacetyltransferase induction by ( 0-isoproterenol or dibutyryl cyclic AMP. The incorporation of labeled amino acids into gland protein was essentially linear over a 6-h experimental period. Examination of the newly synthesized proteins by polyacrylamide gel electrophoresis and autoradiography did not reveal the appearance nor the disappearance of any specific protein(s) caused by (I)-isoproterenol or (I)-propranolol. The lack of stimulation of synthesis of any specific protein was further demonstrated by constant ratio of incorporation in double-label experiments. Either ~FM (I)-isoproterenol or 1 mre dibutyryl cyclic AMP stimulated protein synthesis 20-40s. This increase was not due to an enhanced uptake of precursor radiolabeled amino acids by the glands when incubated with the P-agonist or cyclic AMP derivative. The stimulation of protein synthesis caused by (&isoproterenol was abolished by the P-antagonist (I)-propranolol. These results suggest that P-agonists may increase pineaf gland protein synthesis through their relevant receptor and the generation of cyclic AMP. This increase in synthesis appears to be general and no selective elevation increase in any one band was observed.

In the pineal gland of rats the activity of serotonin N-acetyltransferase (acetyl CoA:serotonin N-acetyltransferase; EC 2.3.1.5) is greatly enhanced by increased sympathetic nerve impulse flow at night (1, 2). The rise in the activity of this enzyme facilitates the production of the pineal hormone melatonin (3-5). The binding of norepinephrine to P-receptors of the pineal gland is thought to stimulate adenylate cyclase and the resultant rise in the cellular 3’:5’-cyclic AMP concentration presumably leads to a 50- to 7O-fold increase in N-acetyltransferase activity (6, 7). The biochemical changes occurring between the rise and fall of cyclic AMP and the rise in enzyme activity a few hours later have not been adequately defined. The stimulation of melatonin production elicited by norepinephrine is inhibited by cycloheximide (8). The effect of cyclohexi-

mide on melatonin production by either norepinephrine or dibutryl cyclic AMP was found to be due to an inhibition in N-acetyltransferase induction suggesting that de nova protein synthesis was required during the induction process (9). This observation was later confirmed and extended to demonstrate that protein synthesis is always required for an increase in pineal gland Nacetyltransferase activity while new ribonucleic acid synthesis may not always be necessary for enzyme induction (10). A previous investigation had utilized radiolabeled tryptophan, methionine, leucine, and an amino acid mix (11) to monitor pineal gland protein synthesis. Other studies have measured the incorporation of labeled leucine (12) to assess the effect of ouabain and elevated potassium ion concentration on gland physiology or used labeled leucine and histidine (13, 14) to characterize the effect of 2-fluoro-1-histidine on pineal gland enzyme induction. While all these prior studies had measured the incorporation of radiolabeled precursor amino

’ Present address: Veterans Administration Hospital, Calcium Research Laboratory, Kansas City, Missouri 64128. I

0003-9861/78/1911-0001$02.00/0 Copyright 0 1978 by Academic Press. All rights of reproduction in any form

Inc. reserved.

2

MORRISSEY

AND

acids into pineal protein, no attempt has been made to determine if any specific proteins are selectively increased during the apparent induction of the enzyme. The present study described protein synthesis in the cultured rat pineal gland during the induction of serotonin iV-acetyltransferase by (1)-isoproterenol and dibutyryl cyclic AMP. MATERIALS

AND

METHODS

Animals. Male-Sprague-Dawley rata (X0-175 g) were housed under diurnal lighting conditions (12 h light, 0600-1800 h) for 1 week prior to experimental use. Animals were exposed to light for 24‘h before sacrifke. Pineal gland culture. Animals were decapitated, and the pineal glands were removed and cultured as described by Deguchi and Axelrod (15). Six to eight pineals were incubated per dish in 2.5 ml of medium at 35’C under an atmosphere of 95% OZ-5% Con. Drugs and isotopes were added to the medium at the times and concentrations indicated.

LOVENBERG bovine serum albumin 68,ooO, chymotrypsinogen 25,Ooo; and ribonuclease 13,700) were electrophoresed in adjacent slots. Proteins were stained with Coomassie brilliant blue and the gel was dried and autoradiographed. N-Acetyltransferase assay. N-Acetyltransferase activity was measured in pineal gland homogenates (equivalent to 0.25 gland) or whole glands by the method of Deguchi and Axelrod (17) with the exceptiol: that the acetyl-CoA concentration was 20 nmol/70 $ incubation and contained 0.1 &i of 1-[14C] acetyl-CoA (New England Nuclear NEC-313, 55 mCi/mmole). Pharmacologic treatment. Pineal glands were incubated in media containing the relevant radioisotope bitartrate (Sigma) or + 1 mM f 2 pM (I)-isoproterenol dibutyryl cyclic AMP (Sigma). When present (&propranolol (Ayrest Laboratories) was used at a final concentration of 10 CM. Stock solutions of these compounds in water were made fresh each day and were added as l/100 of the fmal medium volume. RESULTS

Measurement of pineal gland protein syn. thesis. Pineal glands were incubated in media contain-

Incorporation of Methionine Protein Bands

into Specific

ing radioactive amino acids. The isotopes were obtained from New England Nuclear Corp. and were added in the following amounts for various experiments: “C-amino acid mixture (NEC 445) either 5 or 15 pCi/ml; ‘H-amino acid mixture (NET), 20 yCi/ml, C3H]tryptophan (NET-276, 8 Ci/mmole), 20 &i/ml, or [?S]methionine (NEG-OOSH, 440 Ci/mmole), 5 @/ml. The time of incubation is included for each experiment. Glands were removed from culture individually, rinsed 3 times in 5 ml of physiological saline and homogenized in 0.5 ml of 50 mM sodium phosphate with 1 ml glass-Teflon homogenizers. For the measurement of protein synthesis, the homogenate was mixed with 0.5 ml of 20% trichloroacetic acid and boiled for 10 mm. The samples were cooled on ice, filtered and washed with 10% trichloroacetic acid. The filters were dissolved in lo-ml Instabrays (Yorktown Research) and radioactivity measured in a liquid scintillation spectrophotometer. The radiolabeled free amino acid pool was determined by measurement of the radioactivity contained in the initial trichloroacetic acid supematant of the washed homogenized glands before boiling. Glands which had been incubated with [?S]methionine were pooled and homogenized aa above. A portion of the homogenate was immediately removed for N-acetyltransferase determinations (see below). The remainder of each homogenate was immediately made 1% with respect to sodium dodecyl sulfate and 2-mercaptoethanol and boiled for 2 min. The samples were applied to individual slots of a 10% acrylamide slab gel containing sodium dodecyl sulfate and electrophoresed (16). Marker proteins (phosphorylase 9O,ooO,

Preliminary experiments established that the rate of incorporation of [35S]methionine was linear during the course of a 6-h culture incubation. The specificity with respect to the type(s) of protein synthesized in cultured glands was first examined. Glands were incubated in the presence or absence of 2 PM ( I)-isoproterenol for 6 h in medium supplemented with 5 &i/ml[35S]methionine. The glands incubated with P-agonist incorporated 18% more methionine into protein than the controls (data not shown). Klein’s laboratory has made a similar observation (13, 14). After 6 h, some of the glands which were incubated with the (Z)isoproterenol received (Z)-propranolol at a final concentration of 10 PM for an additional ‘/z h. At the end of the incubation period, the glands were homogenized and an aliquot was removed for N-acetyltransferase activity. The remainder of the homogenate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis in order to separate the newly synthesized radiolabeled proteins by molecular weight. The gel was stained, dried and autoradiographed (Fig. 1). The addition of 2 PM ( I)isoproterenol caused an approximate 25fold increase in IV-acetyltransferase activity but no apparent difference in the pattern of

PINEAL roterend mlolol Activity

12

GLAND

PROTEIN

+ 295

79

-90000 -68GiM

-25cal

Cl

3700

FIG. 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radiolabeled pineal gland protein. Pineal glands were incubated in standard media containing 5 $i/ml[35S]methionine f 2 pM (ll-isoproterenol. After 6 h, some glands received 10 pM (&propranolol. All glands were incubated a total of 6?4 h. Glands were homogenized, N-acetyltransferase activity and gel electrophoresis were performed as described under Materials and Methods. The N-acetyltransferase activity is expressed as picomoles of Nacetyltryptamine formed per 0.25 gland per lo-mm incubation.

3

SYNTHESIS

combined, homogenized and the proteins extracted as described below. As seen in Fig. 2, similar pattern of incorporation was seen for each isotope and no significant differences in 3H/‘4C were observed. Thus even though numerous newly synthesized protein bands were observed no selective incorporation could be detected in the induced glands The Effect of (I)-Isoproterenol Protein Synthesis

on Total

To explore further the apparent stimulation of amino acid incorporation by isoproteol observed above and reported previously (13,14), the rate of incorporation as a function of time of incubation was explored. The addition of 2 PM (&isoproterno1 to the incubation media causes an increase in the incorporation of radioactive amino acids into pineal gland protein compared with similar cultures which did not

Km9x-

synthesized protein. Similarly, while the addition of (Z)-propranolol to the j%agonist induced pineal glands caused a 70% decrease in the N-acetyltransferase activity, there was no change in the radiolabeled protein pattern. Incorporation in Induced

of 3H- and ‘*C-Amino and Control Glands

Acids

A more quantitative approach to detection of a single selectively synthesized protein is to measure isotope ratios from a disc gel in a double label experiment. In these experiments control glands were incubated in the presence of 3H-amino acid mixture (20 @/ml) and stimulated glands in the presence of **C-amino acid mixture (15 &i/ml). At the conclusion the glands were

FIG. 2. Newly synthesized proteins of pineal gland cultured in the absence (cec1) and presence (C-0) of (I)-isoproterenol. The iaoproterenol treated glands were incubated with “C-amino acid mixture 15 &i/ml and control glands incubated with 3H-amino acid mixture. The newly synthesized proteins were extracted and processed as described in the text. Following electrophoresis, the gel was sliced, eluted with 0.5 ml of 0.1% sodium dodecyl sulfate and suspended in 10 ml of Instabrays.

4

MORRISSEY

AND

receive P-agonist (Fig. 3). This increase is rather modest during the first 3 h of gland culture but is significant by 4% h (P < 0.05). Total protein synthesis was increased an average of 22% above the control level by the presence of (I)-isoproterenol. It is also clear that the incorporation of labelled amino acid into pineal gland protein is essentially linear over the 6-h experimental period. If (I)-isoproterenol is exerting its effect on protein synthesis by binding to a receptor and generating cyclic AMP, then this effect should be mimicked by dibutyryl cyclic AMP. Incubation of pineal glands in the presence of ImM dibutyryl cyclic AMP significantly (P < 0.025) increased the incorporation of radioactive amino acids into newly synthesized protein (Fig. 4). This increase is about 40% and is evident at all time points tested through 6 h. The Effect of (I)-Isoproterenol on Amino Acid Uptake A previous investigation (11) had attributed an increase in labeled tryptophan incorporation into pineal gland protein, caused by ,&agonist, to an increase in amino acid uptake by the gland. This observation was made after 2 days of organ culture. Similar uptake enhancements were not observed for leucine or methionine (11) or if 12m,

I

v

I 60

I

1

1

180 240 303 120 MINUTES OF INCUBATION

I 33

FIG. 3. The effect of (Wsoproterenol on pineal gland protein synthesis. Pineal glands were incubated in standard media containing 15 @i/ml ‘%unino acid mixture + 2 pM (I)-isoproterenol for various lengths of time. Glands were removed and processed as described under Materials and Methods. Data represent the mean f SEM for 13 determinations.

LOVENBERG

FIG. 4. The effect of dibutyryl cyclic AMP on pineal gland protein synthesis. Pineal glands were incubated in standard media containing 15 &i/ml %amino acid mixture + 1 miu dibutyryl cyclic AMP for various lengths of time. Glands were removed and processed as described under Materials and Methods. Data represent the mean k SEM for 6 determinations.

dibutyryl cyclic AMP was used (18). We also examined the possibility that the increased incorporation of radioactive amino acids into pineal gland protein was due to an increase in the uptake to radioactive amino acid precursors in the presence of P-agonist or dibutyryl cyclic AMP. At the end of the culture period, glands were washed, homogenized and the trichloroacetic acid soluble radioactivity was determined (Table IA). It is readily seen that while 2 PM (&isoproterenol or 1 mu dibutyryl cyclic AMP caused an approximate 32% increase in radioactive amino acid incorporation into protein, there was no apparent increase in the intracellular pool of free amino acids. Similarly, there was a 15% increase in the labeled tryptophan incorporation caused by 2 PM (I)-isoproterenol and a 24% increase caused by 1 mM dibutyryl cyclic AMP with no increase in the free labeled tryptophan content of the gland (Table IB). If ( I)-isoproterenol is exerting its action through a P-receptor, then the effect of the P-agonist on glandular protein synthesis should theoretically be abolished by /3-antagonists. This was found to be the case (Table I). The increase in both general amino acid and tryptophan

PINEAL

GLAND

PROTEIN TABLE

5

SYNTHESIS

I

THEEFFECTOF(Z-ISOPROTERENOLAND(~)-PROPRANOLOLONPINEALPROTEINSYNTHESIS" (A) Condition

Control + (I)-Isoproterenol (2 PM) + (I)-Isoproterenol (2 pM) and (l)-propranolol (10 PM) + Dibutyryl cyclic AMP (1 mM) + Dibutyryl cyclic AMP (1 mM) (I)-propranolol (10 PM)

and

(B) Condition

Control + (&ISOprOterenOl (2 pM) + (l)-Isoproterenol (2 FM) and (I)-propranolol (10 PM) + Dibutyryl cyclic AMP (1 mM) + Dibutyryl cyclic AMP (1 mM) and (Z)-propranolol (10 FM)

Free amino acids

Amino acids incorporated into protein

N-acetyltransferase activity

19,449 k 3519 19,025 k 1730 17,337 -c 2467’

27,992 + 1491 36,110 + 4053’ 28,762 + 4685

36 * 10 745 + mob 70 + 25

20,059 + 2652 18,469 f 2327

38,759 k 2533’ 37,429 + 41136

825 k 130b 795 + 90*

Free tryptophan

Tryptophan incorporated into protein

43,734 k 4613 42,158 f 4627 42,109 f 8967

47,463 + 3315 54,500 f 8779’ 46,193 + 7870

42,207 + 4302 44,102 f 4527

59,291 -+ 3190h 58,211 + 6312’

(2Pineal glands were incubated for 6 h in media containing 5 $X/ml ‘%-amino acid mixture and 20 pCi/ml [3H]tryptophan and the additions listed above. Glands were homogenized and the labeled amino acid content of the trichloroacetic acid soluble free amino acids or the acid insoluble protein or N-acetyltransferase activity was determined as described under Materials and Methods. Data represents the mean -t SEM of six determinations and are expressed as counts/min/pineal or picomoles of N-acetyltryptamine formed/gland/IO min. h P <0.005. c P
incorporation into protein or N-acetyltransferase induction elicited by 2 PM (Z)-isoproterenol is abolished by 10 j.&M (Z)-proprano101. The increase in pineal gland protein synthesis or IV-acetyltransferase induction caused by dibutyryl cyclic AMP, however, is not affected by the P-antagonist. DISCUSSION

These results suggest that the rate of pineal gland protein synthesis may be subtly regulated by /3-agonists. This may be initiated by the rate of norepinephrine release from sympathetic nerves impinging upon the pineal gland in uiuo (1,19,20), the injection of isoproterenol in uiuo (21) or the addition of ,&agonist to culture media containing glands in vitro (5, 22). These compounds combine with a specific receptor on the pineal membrane, functionally associated with adenylate cyclase, and increase the cellular concentration of cyclic AMP (6, 7). Cyclic AMP has been implicated as a regulator of protein synthesis in a number of eukaryotic systems (23) and presumably

acts through a protein kinase (24). The pineal gland is a rich source of protein kinase (25) and differences in the protein kinase activity of the pineal gland have been observed between supersensitive and subsensitive states (26, 27) or upon P-agonist treatment of glands in culture (28). Treatment of cultured pineal glands with ( I)-isoproterenol or dibutyryl cyclic AMP was found to stimulate the incorporation of labeled amino acids into protein 20-40s. The effect of (I)-isoproterenol on protein synthesis was blocked by ( 1)-propranolol suggesting that a P-receptor was involved in the process. Earlier studies had observed a 15% stimulation of cultured pineal gland protein synthesis, using a similar labeled ammo acid mixture as was used here, caused by ( I)-norepinephrine (11) or 15-36s increase in labeled leucine and histidine incorporation elicited by (Z)-isoproterenol (13, 14). No increase in the incorporation of labeled leucine or methionine by (I)-norepinephrine was found by other workers, however (11). The apparent in-

6

MORRISSEY

AND

crease in labeled tryptophan incorporation into protein by (Z)-norepinephrine was found previously to be due to an enhanced uptake of the radiolabeled amino acid after 48 h of culture but the uptake of the labeled amino acid mixture was not studied (11). Norepinephrine was found to increase the uptake of the model amino acid a-aminoisobutyric acid within 20 min of P-agonist addition but no uptake enhancement was observed at 60 min (11). Our results, based upon 6-h incubation periods for both the mixture of labeled amino acids (15) amino acids not containing tryptophan, methionine, cysteine, asparagine, or glutamine) or for labeled tryptophan, indicate that the increase in amino acid incorporation caused by either (I)-isoproterenol or dibutyryl cyclic AMP was not due to an increase in labeled precursor amino acid uptake by the cultured gland. The stimulation of glandular protein synthesis and N-acetyltransferase induction by dibutryryl cyclic AMP was not inhibited by (I)-propranolol. This is in keeping with the observations of a lack of effect of propranolol on dibutyryl cyclic AMP stimulated production of melatonin (29) or on N-acetyltransferase induction (30, 31). The manner in which P-agonist may control the rate of general protein synthesis in the pineal gland is not known. Protein synthesis in rabbit reticulocytes is regulated by the phosphorylation of a subunit of initiation factor eIF-2 by a cyclic AMP-independent protein kinase (32-35). The activity of the cyclic nucleotide-independent kinase is in turn regulated by cyclic AMP-dependent protein kinase (36, 37). Phosphorylation of the initiation factor, however, leads to an inhibition of reticulocyte protein synthesis, (32-37). Glucagon stimulates the phosphorylation of a rat liver ribosomal protein (38, 39) which has been identified as a small subunit structural protein S6 (40). This same protein is phosphorylated during liver regeneration and it has been speculated that it may relate to a mechanism leading to increased general protein synthesis in the regenerating liver (41,42). The increase protein synthetic capacity of the pineal gland elicited by P-agonist is not expressed as the appearance of any new major protein. The accelerated rate of pro-

LOVENBERG

tein synthesis seems to be general in nature and not associated with any particular protein(s) but rather an apparent uniform increase in the synthesis of all the pineal gland proteins. The addition of (Z)-propran0101 to cultured glands containing high levels of ,&agonist-induced N-acetyltransferase mimics (20, 43) the rapid light-induced inactivation of enzyme observed in uiuo (20, 31). In our experiments, ( Z)-propranolol caused a 70% decrease in enzyme activity, however, there was no alteration of the labeled protein pattern. The inducible pineal gland N-acetyltransferase has a molecular weight of 39,000 with a suspected monomer weight of approximately 10,000 (44). There are no observed changes in the pattern of newly synthesized proteins in these regions of the gel regardless of the experimental conditions. The current experiments which fail to demonstrate a selective increase in any specific protein band, are consistent with the concept that post-translational events account for a significant proportion of the apparent induction of N-acetyltransferase. If, however, N-acetyltransferase is a minor protein component that has an extremely rapid turnover then a small increase in synthesis rate might lead to the significant accumulation of active enzyme if the degradation rate were held constant. In any case it appears that the regulation of Nacetyltransferase activity in pineal gland is more complicated than simple regulation of synthesis rate. REFERENCES 1. KLEIN, D. C., WELLER, J. L., AND MOORE, R. Y. (1971) hoc. Nut. Acad. Sci. USA 68,3107-3110. 2. BROWNSTEIN, M., AND AXELROD, J. (1974) Science 183, 163-165. 3. KLEIN, D. C., AND WELLER, J. (1970) Fed. Proc. 29, 615. 4. KLEIN, D. C., AND WELLER, J. (1970) Science 169, 1093-1095. 5. KLEIN, D. C., BERG, G. R., AND WELLER, J. (1970) Science 168,979-980. 6. STRADA, J. J., KLEIN, D. C., WELLER, J., AND WEISS, B. (1972) Endocrinology 90, 1470-1475. 7. DECUCHI, T. (1973) Mol. Pharmacol. 9, 184-190. a. A~ELROD, J., SHEIN, H. M., AND WURTMAN, R. J. (1969) Proc Nat. Acad. Sci. USA 69, 544-549. 9. KLEIN, D. C., AND BERG, G. R. (1970) Advan. Biochem. Psychophannacol. 3,241-263. 10. ROMERO, J. A., ZATZ, M., AND AXELROD, J. (1975)

PINEAL

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PROTEIN

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