d -Aspartate is present in human retinoblastoma Y79 cells

Neuroscience Letters 267 (1999) 37±40

D-Aspartate

is present in human retinoblastoma Y79 cells

Mitsuko Hayashi, Toshie Itabashi, Yoshinori Moriyama* Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700±8530, Japan Received 25 March 1999; accepted 31 March 1999

Abstract In mammals, D-aspartate is present in various neuroendocrine cells, being especially abundant in pinealocytes. Although D-aspartate is suggested to be involved in some neuroendocrine function, little is known about its origins as well as its physiological roles. In the present study, we found that an appreciable amount of D-aspartate (50.8 pmol=1 £ 106 cells) is present in clonal human retinoblastoma Y79 cells. The amount of D-aspartate corresponds to 28% of that in rat pinealocytes. The D-aspartate concentration did not change with the culture duration or passage, suggesting de novo biosynthesis of it. Thus, Y79 cells may constitute a suitable experimental system for studies on the biogenesis and signal transduction of D-aspartate in mammalian cells. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: D-Aspartate, Retinoblastoma; Y79 cell; Retina; Pinealocyte

Recently, substantial levels of d-amino acids have been detected in mammalian tissues, although little is known about their origin or their physiological functions ([2,5] and reviewed in Ref. [6]). d-Aspartate has been shown to be concentrated in neuronal and endocrine cells, and its role in some neuronal or endocrine function has been suggested [4,15]. Mammalian pinealocytes, endocrine cells for melatonin, contain the highest level of d-aspartate in their cytoplasm [8,17], which is due to de novo biosynthesis [9]. Internal d-aspartate is released from pinealocytes and may inhibit norepinephrine-stimulated melatonin synthesis through an inhibitory cyclic AMP cascade [9]. Thus, pinealocytes provide an unique experimental system for studies on the mechanism of biogenesis of d-aspartate and its mode of action. However, unavailability of pineal clonal cell lines limits biochemical and cell biological studies. To develop more suitable experimental systems, we have looked for a cell line that contains d-aspartate and found that pheochromocytoma PC 12 cells do [12]. However, the content of d-aspartate in PC 12 cells is low, i.e. only 2% of that of pinealocytes. Cell lines containing higher amounts of d-aspartate are needed for quantitative studies. Here, we report that retinoblastoma Y79 cells, a cell line derived from human retinal cells [1,10,13], contain an appreciable amount of d-aspartate. * Corresponding author. Tel.: 181-86-251-7933; fax: 181-86251-7933. E-mail address: [email protected] (Y. Moriyama)

Y79 cells from the American Type Culture Collection (Rockville, MD) were maintained in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 55 mg/ml sodium pyruvate, 6 mg/ml glucose, 0.1 mg/ml streptomycin, 100 units/ml penicillin G and 0.25 mg/ml fungizone at 378C under 5% CO2. a TC6, a clonal a cell line [3], was kindly provided by Dr. K. Hamaguchi (Oita University Medical School, Japan) and maintained in the medium as described [3]. Pinealocytes were isolated from rats (Wistar, male, 3 postnatal weeks) and cultured as described previously [16]. To quantify d-aspartate, the cultured cells (2:5 £ 106 cells) were homogenized in 0.5 ml of ice-cold methanol. The homogenate was centrifuged at 18 000 £ g for 10 min, and the resultant supernatant was evaporated in vacuo, and the resultant residue was dissolved in 20 ml of 50 mM borate buffer (pH 8.0) and 30 ml of 50 mM 4-¯uoro7-nitro-2,1,3-benzoxadiazole (NBD-F) in methanol. It was heated at 608C for 5 min. Then, 50 ml of 1% tri¯uoroacetate in methanol was added. The reaction mixture was ®ltered through a membrane ®lter (pore size, 0.5 mm) (Advantec Dismic-03 CP ®lter; Toyo Roshi, Japan), and then subjected to reversed phase high pressure liquid chromatography (HPLC) to obtain the fraction containing d-aspartate. Then, the d-aspartate-containing fraction was evaporated to dryness in vacuo, and the resultant residue was dissolved in 3% acetate in methanol. Then, aliquots (20 ml) were analyzed by HPLC on a Sumichiral OA-2500 (R) column as described [8,9,12]. Isocratic elution was performed with 4

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 31 4- 6

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M. Hayashi et al. / Neuroscience Letters 267 (1999) 37±40

Fig. 1. Identi®cation of D-aspartate in Y79 cells on HPLC. (A) Standard D- and L-aspartate, 250 pmol each, were applied. (B) Detection of Daspartate in Y79 cells. (C) The same sample (150 ml) in (B) was evaporated to dryness in vacuo, and then the resultant residue was suspended in 20 ml of 50 mM borate buffer (pH 8.0) containing D-amino acid oxidase from porcine kidney (0.03 units) (Type I, Sigma). After incubation at 378C for 1 h, the amount of D-aspartate was determined. (D) Absence of D-aspartate in a TC6 cells. The positions of Land D-aspartate are indicated by arrows.

mM citric acid in methanol as the mobile phase at the ¯ow rate of 1.0 ml/min. The ¯uorescence of NBD-amino acids was detected at 530 nm with an excitation wavelength of 470 nm. To measure melatonin, Y79 cells and pinealocytes were maintained for 5 days, washed with the above medium, and then cultured further for 1 day. Then, 1 mM d-aspartate was added to the medium in the presence or absence of 20 mM forskolin [10,13]. After further incubation for 6 h, the medium and cells were carefully separated and the melatonin content was measured as described [14]. Polyclonal antibodies against d-aspartate were raised in rabbits through successive injections of emulsi®ed d-aspartate conjugated with bovine serum albumin with glutaraldehyde (5 mg each) every 2 weeks for 2 months, as described by Lee et al. [11]. The site-speci®c antibodies were used after puri®cation on an af®nity column of d-aspartate immobilized on cyanogen bromide-activated Sepharose 4B (Pharmacia). The speci®city of the antibodies to d-aspartate has been reported elsewhere [11,15]. To identify d-aspartate immunohistochemically, cells on poly L-lysine-coated glass coverslips were ®xed in 4% paraformaldehyde for 20 min, washed with phosphate-buffered saline (PBS), incubated with PBS containing 0.1% Triton X-100 for 30 min, and then further with 10% goat serum in PBS, and ®nally reacted with the antibodies at 1 mg/ml in PBS containing 2% goat serum for 1 h. The samples were washed three times with PBS and reacted with the second antibodies conjugated with ¯uorescein-isothiocyanate, and ®nally the immunoreactivity was observed under an Olympus BH-2 ¯uorescence microscope [7]. HPLC analysis on a Pirkle-type chiral column demonstrated the presence of d-aspartate in Y79 cells, i.e. 50.8 pmol=1 £ 106 cells (Fig. 1). This amount of d-aspartate corresponds to 3.5% of the total free aspartate, and is about 28% of that in pinealocytes [8], and about 14-fold that in PC12 cells [12]. The peak of d-aspartate disappeared upon incubation with 0.03 units of d-amino acid oxidase

(Type I, Sigma) (Fig. 1C). The d-aspartate peak was not observed for the other cell lines examined, such as a TC6 cells (Fig. 1D). Upon treatment with digitonin at 50 mM, about 70% of the d-aspartate in Y79 cells was recovered in the culture medium, suggesting cytoplasmic localization of the amino acid in the cells. The presence of d-aspartate in Y79 cells was also demonstrated by means of immunohistochemical techniques. As shown in Fig. 2, strong d-aspartate immunoreactivity was observed in all Y79 cells and pinealocytes examined, but not in a TC6 cells. The immunoreactivity in these cells is uniform throughout the cells, again supporting the cytoplasmic localization of d-aspartate in these cells. The content of d-aspartate in Y79 cells is independent of the culture duration and the passage number: essentially the same amounts of d-aspartate per 1 £ 106 cells were detected on culture for 2 and 7 days and in cells with two and ®ve passages. Since the d-aspartate content in the medium was less than 1 fmol, these results suggest that d-aspartate is not of exogenous origin and support the occurrence of de novo biosynthesis of d-aspartate in the cells. Y79 cells are the only clonal cell line that maintains the ability of melatonin synthesis [10,13]. Upon stimulation by forskolin, the cells produced 3.5 pmol of melatonin per 1 £ 106 cells, which is consistent with the previous reports [10,13] (Fig. 3). We examined whether or not exogenous d-aspartate affects the forskolin-stimulated melatonin synthesis, since it inhibits melatonin synthesis in pinealocytes [9]. In contrast to in pinealocytes, however, 1 mM daspartate did not affect the melatonin synthesis in Y79 cells, whereas it inhibited 80% of the melatonin synthesis in pinealocytes under similar assay conditions (Fig. 3). Thus, the response of Y79 cells to d-aspartate differed from that of pinealocytes. Here, we report that Y79 cells contain an appreciable amount of d-aspartate, possibly due to de novo biosynthesis. It is noteworthy that both Y79 cells and pinealocytes

M. Hayashi et al. / Neuroscience Letters 267 (1999) 37±40

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Fig. 2. Immunohistochemical detection of D-asparate in Y79 cells and rat pinealocytes. Cultured Y79 cells (A,B), rat pinealocytes (C,D), or a TC6 cells (E,F) were stained immunohistochemically with anti-D-aspartate antibodies, and then observed under a Nomarsky microscope (A,C,E), or a ¯uorescence microscope (B,D,F). Scale bar, 10 mm.

produce melatonin and contain high concentration of daspartate in common. Although no interrelationship between the abilities of melatonin and d-aspartate synthesis in these cells is known at present, we supposed that d-aspartate is somehow involved in melatonin synthesis as in pine-

alocytes. In pinealocytes, d-aspartate stimulates the intrinsic glutaminergic system recently identi®ed [16], and inhibits melatonin synthesis through an inhibitory cAMP cascade via metabotropic glutamate receptor type 3 ([9], and Ishio and Moriyama, in preparation). The absence of a response to

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M. Hayashi et al. / Neuroscience Letters 267 (1999) 37±40

Fig. 3. The effects of D-aspartate on melatonin synthesis in Y79 cells and pinealocytes. Typical patterns of separation of melatonin were shown: standard melatonin (20 pmol) (A) Y79 cells in the absence (B) or presence of forskolin (C,D), and pineal gland in the presence of forskolin (E,F). D-Asparate at 1 mM was also included in the assay medium (D,F). The melatonin peak is indicated by arrows.

the amino acid in melatonin synthesis might be attributed to the absence of this class of receptor or a lack of parts of the signaling cascade in Y79 cells. We have now extensively studied the expression of functional glutamate receptors and the protein factors involved in the signaling pathways in Y79 cells. In conclusion, Y79 cells may constitute a suitable experimental system for the biogenesis and signal transduction of d-aspartate. Comparison of the effect of d-aspartate in Y79 cells with that in pinealocytes is useful for understanding the molecular mechanism of the d-aspartate-evoked signaling cascade. We are grateful to Dr. H. Yamada for his help in the measurement of d-aspartate and melatonin. This work was supported, in part, by grants from the Japanese Ministry of Education, Science and Culture, the Salt Science Foundation and the Terumo Science Foundation. M.H. was supported by the Hayashi Memorial Foundation for Female Natural Scientists. [1] Besharse, J.C., Iuvone, P.M. and Pierce, M.E., Regulation of rhythmic photoreceptor metabolism: a role for post-receptoral neurons. Prog. Retinal Res., 7 (1988) 21±61. [2] Dunlop, D.S., Neidle, A., McHale, D., Dunlop, D.M. and Lajtha, A., The presence of free d-aspartic acid in rodents and man. Biochem. Biophys. Res. Commun., 141 (1986) 27± 32. [3] Hamaguchi, K. and Leiter, E.H., Comparison of cytokine effects on mouse pancreatic a-cell and b-cell lines. Diabetes, 39 (1990) 415±425.

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