Retinoic acid treatment enhances the acetylcholine contents in the human teratocarcinoma cell line NTera-2

Regulatory Peptides 96 (2000) 59–63 www.elsevier.com / locate / regpep

Retinoic acid treatment enhances the acetylcholine contents in the human teratocarcinoma cell line NTera-2 Hanspeter S. Fischer, Irene Berti, Dieter S. Schatz, Christian Humpel, Alois Saria* Division of Neurochemistry, Department of Psychiatry, University Hospital Innsbruck, Anichstrasse 35, A-6020, Innsbruck, Austria

Abstract Human NTera-2 / clone D1 teratocarcinoma cells are induced by retinoic acid (RA) to differentiate into postmitotic cells with morphological and biochemical characteristics of embryonic human neurones. Currently only limited information concerning peptidecontents and neurotransmitter pools of these cells is available. Zeller and Strauss [Int. J. Dev. Neurosci. 1995;13(5):437] described an increase in choline acetyltransferase (ChAT) activity in RA-treated, but not in untreated NTera-2 cells, suggesting the induction of a cholinergic phenotype during treatment with RA. In the present study we investigated the effect of RA-differentiation on the amount of the neurotransmitters acetylcholine (ACh), and dopamine in NTera-2 in order to specify the transmitter phenotype induced by RA-differentiation. We found that a 4-week treatment of NTera-2 cells with 10 mM RA markedly increased the ACh-content of these cells, while dopamine levels were unchanged. Depolarisation with potassium (60 mM) enhanced ACh-outflow in the differentiated cells in a Ca 11 dependent way. Also neuropeptides like substance P and NPY were detectable in the undifferentiated NTera-2 cells, while vasointestinal peptide (VIP) could not be found in either precursor or RA-differentiated cells. Differentiation was accompanied by a marked reduction of neutral endopeptidase enzyme activity and aminopeptidase activity. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of this human teratocarcinoma cell line, and that these cells might provide a model system to investigate cholinergic properties of human origin.  2000 Elsevier Science B.V. All rights reserved. Keywords: Retinoic acid; NTera-2; Acetylcholine

1. Introduction The NTera-2 cell line is a human teratocarcinoma cell line which is neuronally committed [2]. Retinoic acid (RA) is a naturally occurring derivative of vitamin A known to be synthesised in brain [3] and to affect neuronal differentiation within the CNS [4]. After differentiation with RA, this cell line expresses typical markers of CNS neurones like neurofilaments [5] tau protein and synaptophysin, microtubule associated proteins (MAP) type 1 and type 2 [6]. Neurones derived from this cell line also express NMDA and non-NMDA glutamate receptor genes [7,8], as well as amyloid precursor protein [9]. Therefore interest has increased in using this human cell line as a model *Corresponding author. Tel.: 1 43-512-504-3710; fax: 1 43-512-5043716. E-mail address: [email protected] (A. Saria).

system for investigation of excitotoxicity and neurodegenerative disorders like Alzheimer’s disease [10]. Despite its good characterisation with respect to biochemical changes and receptor equipment during differentiation, relatively scarce information is available about the neurotransmitters synthesised by this cell line. Recently it has been demonstrated that NTera-2 cells express tyrosine hydroxylase when implanted into the caudatoputamen of mice [11], suggesting the differentiation into a catecholaminergic phenotype. In contrast, when treated with RA, these cells also induce enzymes related to cholinergic functions in brain, like ACh-esterases [12] or the expression of M2 and M3 type muscarinic ACh receptors [13]. Zeller and Strauss [1] reported for the first time that RA treatment enhances the choline acetyltransferase (ChAT) enzyme activity in NTera-2 cells, and thereby provided the first evidence for the induction of a cholinergic phenotype in these cells. An important hallmark of neuronal differen-

0167-0115 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 00 )00201-9

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tiation is the capacity of neurones to synthesise the appropriate neurotransmitters, thereby determining their transmitter phenotype. In the present study we quantified the amount of ACh and dopamine synthesised by NTera-2 cells before and after their differentiation by RA as well as some peptidergic neurotransmitters like substance P, neuropeptide Y (NPY) and vasointestinal peptide (VIP). The aim of the project was to further characterise the transmitter phenotype of these cells in order to improve the applicability of this human cell line for pharmacological research.

2. Materials and methods

acetic acid. Dopamine was extracted in 0.025 mol / l HCl and analyzed electrochemically with an ESA Coulochem detector (ESA, Bedford, MA, USA) as described by Schatz et al. [16]. The neuropeptides substance P, VIP, neurotensin and NPY were quantified by the use of radioimmunassays (RIA). Extraction was performed by homogenising the cells in ice-cold acetic acid (2 N) by the use of a sonifier (Branson Ultrasonics Corporation, CT, USA). Thereafter the samples were lyophilized and redissolved in assay buffer immediately before RIA. Highly specific antisera for substance P, VIP and NPY were obtained from Peninsula Laboratories (CA, USA) and the respective radiolabeled tracers from Amersham International (Buckinghamshire, UK).

2.1. Cell culture 2.3. Determination of peptidase activities NTera2-precursor cells were obtained from Stratagene (Stratagene Ltd., Cambridge, UK). The undifferentiated cells were maintained in T75 culture flasks (Falcon  , Becton Dickinson, NJ, USA) in D-MEM / F12-medium (Gibco, Becton Dickinson, NJ, USA) containing 10% fetal calve serum, 2 mM glutamine and 1% (v / v) penicilline– streptomycin and amphothericin B and split twice a week. The medium was changed three times a week. For differentiation, the cells were seeded at a density of 6 3 10 4 / cm 2 in six-well culture wells (Falcon  ). Differentiation was induced by the addition of 10 mM trans-retinoic acid (Sigma, St. Louis, USA) (in D-MEM / F12-medium, 10% fetal calve serum, 1% (v / v) penicilline–streptomycin). NT2-precursor cells were treated with RA for at least 4 weeks. ACh-release experiments were performed in T25 culture flasks (Falcon). Differentiated cells were incubated for 30 min in 3 ml of Krebs Ringer Buffer (KRB; 378C, pH 7.4, 25 mM HEPES, 18 mM NaCl, 4.6 mM KCl, 1.17 mM MgSO 4 , 2.5 mM CaCl 2 , 1.17 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 0.1% BSA, 10 mM neostigmine) for determination of basal ACh-release, and subsequently stimulated for 30 min with 100 ml of KCl solution (final concentration 60 mM). In Ca 11 -free KRB, Mg 11 was substituted for Ca 11 .

2.2. Determination of neurotransmitters ACh was determined after HPLC-separation and subsequent electrochemical detection via oxidation at a platinum electrode ( 1 500 mV vs. Ag–AgCl 2 ) according to Damsma et al. [14] and modified by Prast et al. [15]. The amount of ACh was quantified using calibration curves of external standards. The identity of the ACh-peak was verified via omission of the enzyme reactor. For measurement of ACh-contents the cells were washed, harvested by scraping and subsequently sonicated in icecold phosphate buffer (pH 7.4) containing 10 mM of the ACh-esterase inhibitor neostigmine. For determination of neuropeptides, cells were harvested in ice cold 0.2 M

For the quantification of specific enzyme activities the cells were homogenised in ice-cold Tris–HCl (50 mM, pH 7.4) and peptidase activities determined as described by Waters et al. [17] (aminopeptidase N, APN) and Selmeci et al. [18] (neutral endopeptidase, NEP). Enzyme activities are expressed as the change in absorbtion of p-nitroanilide / min mg protein. Protein contents were determined by Bradford’s method [19].

2.4. Statistics Data are presented as means6S.E.M. Statistical comparisons were performed with the Student’s t-test. A twotailed P , 0.05 was accepted as statistically significant.

3. Results Treatment of the cells for 5 weeks with 10 mM RA resulted in a series of morphological changes. NTera-2 cells that have never been in contact with RA, showed a morphology of a confluent polygonal cell layer, with an intense cell–cell contact. When incubated in RA-containing medium, the former epithelial-like cells lost their cell–cell contacts and began to develop cytoplasmic processes and formed network-like structures. Also an increasing number of vacuolization could be observed (Fig. 1a and b). The quantification of ACh-tissue contents revealed that in the undifferentiated cells minimal amounts of ACh were detectable aswell. However, in cultures grown for 4 weeks without retinoic acid, the ACh contents did not further increase (not shown). A treatment with RA markedly enhanced the amount of ACh within the first week and was even more pronounced after about 4 weeks (Fig. 2). ACh could be released from the differentiated cells by stimulation with 60 mM K 1 , thereby enhancing basal AChoutflow at about 30%. However, potassium did not elevate

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Fig. 1. Representative photomicrographs of the morphological appearance of NTera-2 cells cultivated for 4 weeks in normal growth medium (A) and grown in differentiation medium containing 10 mM RA (B). Magnification 5 3 200.

Fig. 2. Effect of a continuous 5 week-treatment with 10 mM RA on ACh-contents in NTera-2 cells. Data represent the absolute amounts of ACh (fmol / well)6S.E.M. 11

ACh-release when Ca was omitted in the incubationmedium (Fig. 3). In contrast to ACh no dopamine was detectable in either untreated or RA-differentiated cells. Undifferentiated NTera-2 cells contained detectable amounts of the neuropeptides substance P and NPY. Whereas no VIP-like immunoreactity could be found in either undifferentiated or differentiated cells (Table 1). To obtain information about peptidergic neurotransmission in NTera-2 cells during differentiation, we additionally quantified the specific enzyme activities of the two peptidases neutral endopeptidase (EC 3.4.24.11) and aminopeptidase (EC 3.4.11.2). In cultures treated with RA both peptidaseactivities were significantly reduced when compared to control cultures (Table 1).

4. Discussion The morphological changes induced by RA are paral-

Fig. 3. Effect of a stimulation with 60 mM KCl on ACh-release in NTera-2–cells treated for 4 weeks with 10 mM RA. Data represent the amount of ACh expressed as percent of control in the respective fraction. Filled bars indicate conditions under 2.5 mM Ca 11 , striped bars indicate conditions in Ca 11 free-medium. Each bar represents the mean6S.E.M. of five cultures (*P , 0.05, Student’s paired t-test, two sided).

leled by an increase of ACh-contents in the differentiating NTera-2 cells. These data confirm previous findings of a cholinergic differentiation of NTera-2 carcinoma cells after RA-treatment [1] and strengthens the possible in vivo role of RA in the determination of transmitter phenotype [4]. Zeller and Strauss[1] noticed that specific ChAT-activity was enhanced in unenriched cultures containing differentiated and undifferentiated NTera-2 cells. Consistent with these data no further enrichment of the cultures for NTera2 neuronal cells was required to enhance the ACh-content in these cells, suggesting that trophic factors synthesised by undifferentiated cells might positively influence the cholinergic properties of these cells. Thus, a mixed culture system of differentiated and undifferentiated NTera-2 cells provides an attractive model system for investigating cholinergic properties of neuronal human origin. The fact

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Table 1 Quantitative concentrations of neurotransmitters and enzymes before and after treatment with RAa Untreated NT2 (fmol / well)

After 4 weeks of RA-treatment (fmol / well)

No. of experiments

216.00622 46.7560.85 , 0.27 (n.d.)

not tested not tested , 0.27 (n.d.)

4 4 4

Neurotransmitter Ach Dopamine

1534.006312 , 1950.00 (n.d.)

2513767795 , 1950 (n.d.)

6 3–5

Enzyme activities NEP APN

(dE / min mg protein) 0.06260.005 0.12660.009

(dE / min mg protein) 0.01760.002*** 0.09360.007*

4–5 4–5

Neuropeptide SP NPY VIP

a NTera-2 teratocarcinoma cells were cultured in control medium or for 4 weeks in presence of 10 mM RA. Neurotransmitter levels were determined as described in Section 2. Values are expressed as mean6S.E.M. of 3–6 cultures. *P , 0.05, ***P , 0.001, n.d. 5 not detectable.

that RA-differentiated NTera-2 cells also release ACh after depolarization in a Ca 11 -dependent way points to the exocytotic mechanisms of release and suggests that these cells offer the opportunity to study functional activity of human cholinergic neurones. Treatment of NTera-2 cells with RA for 2 weeks was sufficient to obtain an amount of tissue ACh comparable to that found in cultures of rat basal forebrain neurones [20]. However, we also noticed, that the cells tended to change their morphology spontaneously; in this case new batches had to be used. Thus spontaneous differentiation may occur and therefore care must be taken when using this cell system over a long term period. In our experiments no dopamine could be detected either in the precursor or the RA-treated cells. Since dopamine synthesis is a prerequisite for norepinephrine synthesis [21] NTera-2 cells are unlikely to differentiate into a catecholaminergic phenotype under the investigated conditions. Although differentiated NTera-2 cells express tyrosine hydroxylase, the amount is relatively low unless they are cultivated together with extracts of the caudatoputamen [11]. This suggests that RA specifically stimulates the expression of cholinergic properties in NTera-2 cells, and that ‘differentiation factors’ distinct from RA are required for the induction of a catecholaminergic phenotype. The present data are well in line with previous studies suggesting a role of RA on the transmitter choice of developing neurons. Likewise RA enhances the expression of cholinergic neuronal markers in various neuronal cell systems like cultured sympathetic neurones of superior cervical ganglia [22,23] and clonal rat pheochromocytoma cells (PC12) [24]. In contrast RA reduces the level of dopamine in human neuroblastoma cell line NB69 and fetal rat midbrain neurones [25,26] and down-regulates the key enzymes of catecholamine synthesis tyrosine kinase and dopamine b-hydroxylase [22]. Since neuropeptides like substance P or NPY could be

quantified in undifferentiated NTera-2 cells, these cells also possess the biochemical properties for peptidergic transmission. So far we have not succeeded in determining substance P or NPY in differentiated NTera-2 cells, however, the lack of vasointestinal peptide immunoreactivity in precursor or differentiated cells excludes the synthesis of VIP at either differentiation stage. Also the metabolic pathway for peptide degradation is present in this human teratocarcinoma cell line. The down-regulation of peptidase activities of NEP and APN reflects an alteration of peptidergic transmission during differentiation. Carvalho et al. [27] showed that a thermolysin-like metallo-endopeptidase is also downregulated in a human neuroblastoma cell line SK-N-BE(2) after RA-induced differentiation into a neuronal phenotype. The altered peptide degradation after RA-induced treatment of NTera2 cells therefore not only serves as a marker for NTera-2 cell differentiation, but might also influence trophic and regulatory functional effects of various peptides. In conclusion, the present study demonstrates for the first time, that treatment of NTera-2 with retinoic acid cells enhances ACh-tissue contents of these cells and induces a cholinergic but not a catecholaminergic phenotype. Retinoic acid induced differentiation of NTera-2 cells might provide an appropriate model system to investigate cholinergic properties of human origin as well as mechanisms determining the induction of a cholinergic phenotype during differentiation.

Acknowledgements This study was supported by the Austrian National Bank Funds project 6564 and the Austrian Science foundation grant F00206. The technical help of Mrs Margit Auffinger, Mrs Astrid Saria and Mrs Iris Berger is gratefully acknowledged.

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