Brain Research, 557 (1991) 303-307 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124785Y

303

BRES 24785

Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity Akinori Akaike, Yutaka Tamura, Yuko Sato, Kumi Ozaki, Rika Matsuoka, Setsuko Miura and Tomoko Yoshinaga 2nd Department of Pharmacology, Facultyof Pharmacy and PharmaceuticalSciences, Fukuyama University, Fukuyama (Japan) (Accepted 30 April 1991)

Key words: Cholecystokinin; Primary culture; Glutamate; Neurotoxicity; Cerebral cortex; N-Methyl-D-aspartate

The effects of cholecystokinin (CCK) on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Brief exposure of glutamate followed by an incubation with normal solution for more than 60 rain reduced cell viability by 60-70%, compared with control values. Glutamate-induced neurotoxicity was significantly inhibited by MK-801 and ketamine, which are non-competitive blockers of N-methyl-D-aspartate (NMDA) receptors. Octapeptide CCK-8S and CCK-related decapeptide ceruletide at concentrations of 10-9-10-7 M dose-dependently reduced glutamate-induced neurotoxicity. A desulfated analog CCK-8NS, which acts selectively as an antagonist of CCKe receptors, also reduced glutamate neurotoxicity. The neuroprotective effects of CCK were antagonized by L-365260, a CCKe receptor antagonist, but not by L-364718, a C C K A receptor antagonist. These results suggest that CCK protects cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via CCK. receptors. High concentrations of cholecystokinin (CCK) are found in the neocortex of the rat 1'2 and guinea pig 1~. CCK-like immunoreactivity occurs predominantly in non-pyramidal bipolar cells 15A6. The addition of depolarizing concentrations of K + or glutamate evoked the release of CCK-like immunoreactivity from the cerebral cortex, whereas the addition of G A B A decreased the resting release of CCK-like immunoreactivity24. The iontophoretic application of CCK induced excitation of cortical neurons in vivo 6'19. These observations suggest that CCK may act as an excitatory transmitter in the cerebral cortex. It has been postulated that the neurotoxicity of glutamate plays an important role in the pathogenesis of the neuronal cell loss which is associated with several neurological disease states s. Recent studies using cultured cortical neurons have demonstrated that brief exposure to glutamate produces delayed degeneration in mature cortical cells over the next few hours 7'9. Competitive or non-competitive blockers of the N-methylD-aspartate (NMDA) subclass of glutamate receptors protected neurons from glutamate neurotoxicity in cortical cultures ~°,14. Thus, it has been postulated from these studies that the N M D A receptor is the predominant route of glutamate-induced toxicity in cortical neurons. The object of the present study was to investigate the

role of CCK in the process of neuronal damage in the central nervous system (CNS). For this purpose, the effects of CCK on glutamate-induced neurorial death were assessed using cultured neurons obtained from whole cerebral cortex. Since the predominant form of CCK in the CNS is the sulfated carboxy-terminal octapeptide, CCK-8S 13'2°, CCK-related peptides, including CCK-8S, were used in the present study. Dissociated murine cortical cell cultures were prepared following the method described by Dichter 12, with some modification. Whole cerebral cortex was removed from fetal rats (16-18 day gestation). The tissue was minced, mechanically dissociated using scalpel blades, filtered using stainless steel mesh (150 mesh), and plated as single-cell suspensions on plastic coverglasses which were placed in Falcon 60 mm dishes (3-4.5 x 106 cells/dish) in a plating medium of Eagle's minimal essential medium (MEM, Eagle's salts) supplemented with 10% heatinactivated fetal bovine serum (1-9 days after plating) or 10% heat-inactivated horse serum (10-14 days after plating), glutamin~ (2 mM), glucose (total 11 mM), sodium bicarbonate (24 mM), and H E P E S (10 mM). Cultures were maintained at 37 °C in a humidified 5% CO 2 atmosphere. After 8 days of plating, culture of non-neuronal cells was halted by the addition of 10-5 M cytosine arabinoside. Only mature (10-14 days in vitro)

Correspondence: A. Akaike, 2nd Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama 729-02, Japan.

31)4

cultures were used in the study. Experiments were carried out in Eagle's solution at 37 °C. Glutamate-induced neurotoxicity was quantitatively assessed by the examination of cultures under Hoffman modulation microscopy at x400. After the completion of drug treatment, cell cultures were stained with 0.4% Trypan blue solution at 37 °C for 10 min and were then fixed with isotonic formaldehyde (pH 7.0, 2-4 °C). The fixed cultures were rinsed with physiological saline for microscopic observation. Cells stained by the Trypan blue treatment were regarded as non-viable. More than 200 cells in each coverglass were counted to determine viability of the cell cultures. In each experiment, 3-6 coverglasses were used to obtain mean + S.E.M. of the cell viability. Drug protection against glutamate-induced neurotoxicity was calculated using the following equation:

Protection ( % ) -

D-G - C-G

x 100

in which D is the viability of the cell cultures treated with a drug and glutamate, G is the viability after glutamate treatment and C is the viability of non-treated cultures. These values for each protection rate were determined on sister cultures. The viability of non-treated cells (C) maintained in vitro for 10-14 days was 86.2 + 1.2% (mean + S.E.M., obtained from 24 coverglasses used as control). CCK-octapeptide sulfated form (CCK-8S, Peptide Institute), CCK-octapeptide desulfated form (CCK-8NS, Peptide Institute), and ceruletide diethylamine (Shionogi) were dissolved in distilled water for stock solutions. (-) L-364718 (synthesized at Shionogi) and (+)L-365260 (synthesized at Shionogi) were dissolved in dimethyi sulfoxide (DMSO) at a concentration of 0.4%. The stock solutions of these drugs were kept below -20 °C and diluted with Eagle's solution immediately before the experiments. The final concentration of D M S O added to the culture was 10-4%. D M S O at 10-4% did not affect the viability of the cultures. Ketamine (Sigma), MK-801 (Research Biochemicals), and monosodium glutamate (Nacalai Tesque) were dissolved in Eagle's solution before the experiments. Fig. 1. shows the neurotoxic effects of glutamate (0.1-1 mM) on cortical cell cultures. Cultures were exposed to glutamate-containing solution for 10 min and were then incubated with normal solution. Incubation of the cultures for more than 1 h was needed to observe marked reduction of the viability. The cell viability was dose-dependently reduced when cultures were incubated

in normal solution for more than 1 h (50 min in Eagle's solution + 10 min in dye-containing solution) after glutamate treatment. Since there was no large difference between the values of 1-h incubation and 24-h incubation, drug-induced protection against glutamate neurotoxicity was determined under the following conditions: 10-min exposure to 1 mM glutamate, followed by 1-h incubation with normal solution. CCK-8S at concentrations of 10-~-10 -7 M inhibited glutamate-induced neurotoxicity. Drugs were added to both the glutamate-containing solution and the normal solution for 1-h incubation. Fig. 2 shows an example of CCK-8S-induced protection against glutamate neurotoxicity. CCK-8S at 10 -~ M (Fig. 2C) and 10 v M (Fig. 2D) markedly reduced the number of the cells stained by Trypan blue. Fig. 3 summarizes the effects of the N M D A antagonists and CCK-related peptides. MK-801 and ketamine, channel blockers of N M D A receptors 22, induced significant protection against glutamate toxicity. These drugs were also added to both the glutamate-containing and the normal solutions. Effective concentrations of MK-801 and ketamine were of the order of micromolar and millimolar, respectively. In the case of MK-801, concentrations of more than 10 -6 M were needed to gain more than 50% protection. Ketamine appeared to be much weaker than MK-801. We tested 3 C C K receptor agonists. CCK-8S and ceruletide, the CCK-related decapeptide, have been reported to act as agonists of both C C K A and CCK B

100

t_ u

50

c 0J w L

g

0

i

0 TJme a f t e r Fig.

i

i

i

1

1. G l u t a m a t e - i n d u c e d

_w

2

glutamate

exposure

neurotoxicity

i

24 (h) of cultured

cortical

neu-

Cultures were exposed to glutamate at 0.1 mM (I-q), 0.2 mM (O), 0.5 mM (A) and 1.0 mM (0) for 10 min. Ordinate: viability of the cultures expressed as a percentage of the viability of non-treated cultures. Each symbol and bar show mean and S.E.M., respectively. The points without bars indicate that the S.E.M.s for these data were smaller than the diameters of the symbols. Abscissa: time after the cessation of glutamate exposure. The time shown in this figure was the summation of the incubating periods with both Eagle's solution and dye-containing solution. Since it took 10 min to complete Trypan blue staining, 10 min after glutamate exposure did not include the incubation with Eagle's solution. rons.

305 A

C

B

D

I

Fig. 2. Effects of CCK-8S on glutamate-induced neurotoxicity. Culture fields were photographed after Trypan blue staining followed by formalin futation. Experiments shown in this figure were done on the same day with sister cultures. A: non-treated cells (control). B: glutamate-treated cells. C: cells treated with CCK-8S at 10-s M and glutamate. D: cells treated with CCK-8S at 10-7 M and glutamate. In B-D, cultures were exposed to glutamate (1 mM) followed by incubation with normal solution. Drugs were added to both the glutamate-containing and normal solutions.

receptors 4, whereas CCK-8NS, a desulfated analog of CCK-8S, has b e e n r e p o r t e d to act as a relatively selective agonist of C C K a receptors 17. A l l of these c o m p o u n d s p r o t e c t e d g l u t a m a t e toxicity at concentrations of 10 -s and 10 -7 M. A t 10 -7 M, ceruletide was most effective, but CCK-8S and CCK-8NS also p r o d u c e d protection of m o r e than 70%. Since the C C K n r e c e p t o r agonist, CCK-8NS,

Q ccKiSNS/ ~i / ~ /

100

CLT

#~OCCK

L~ 5 0

8S

• MK-801

TABLE I Ketamine ~]

'

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

o 0

showed protection which was similar in p o t e n c y to that of CCK-8S itself, selective C C K r e c e p t o r antagonists were used to d e t e r m i n e the r e c e p t o r subtypes of C C K receptors relating with neuroprotection. A s shown in Table I, L-364718 (MK-329) 5, a C C K A r e c e p t o r antagonist, did not affect CCK-8S-induced protection against glutamate neurotoxicity. In contrast, L-365260 TM, a C C K B r e c e p t o r antagonist, significantly antagonized the e f f e c ( of CCK•8S.

I 10 - g

I 10.8

I 10 .7

I 10 -6

Drug c o n c e n t r a t i o n

I 10 . 5

10 - 4

10. 3

(M)

Fig. 3. Dose-response relationships of the neuroprotective effects of cholecystokinin-related compounds and NMDA antagonists. Protection (%) was calculated using the equation shown in the text. CLT, ceruletide.

Effects of L.364718 (CCKA receptor antagonisO and L-365260 (CCKB receptor antagonis 0 on CCK-induced protection against glutamate neurotoxicity Treatment

n

Viability (%)

Control (non-treated) Glutamate (1 mM) Glutamate + CCK-8S (10-7 M) L-364718 (10-s M) + CCK-8S L-365260 (10-s M) + CCK-8S

5 5

90.5 + 1.5 28.2 + 8.0

5 5 5

73.8 _ 2.4 64.7 + 2.3 25.0 + 4.4**

**P < 0.01 (Dunnett two-tailed test, compared with CCK-8S).

306 It has been r e p o r t e d that glutamate-induced neurotoxicity in cortical cell culture consisted of two actions, i.e., the fast swelling induced by an influx of Na ÷ and the delayed injury induced by an influx of Ca 2÷. The fast swelling is a reversible p h e n o m e n o n , whereas the delayed injury or cell death is an irreversible p h e n o m e n o n 8. Choi et al. 9 have d e m o n s t r a t e d that N M D A r e c e p t o r - m e d i a t e d

C C K A receptors and C C K B receptors. C C K A receptors have been considered to be p e r i p h e r a l in type, although they are also distributed in the CNS. Electrophysiological studies have d e m o n s t r a t e d that C C K p r o d u c e d excitation of neurons in a wide region of the central nervous system including neocortex 6"19, h i p p o c a m p u s 3, and nucleus accumbens 21'23. The excitatory effects of C C K on the

neurotoxicity is a m a j o r cause of delayed cell death after brief exposure to glutamate 1°'14. Therefore, the present study aimed to d e t e r m i n e the effect of C C K r e c e p t o r agonists on the delayed neuronal death induced by glutamate. CCK-8S and other CCK-derivatives p r o t e c t e d against glutamate-induced neurotoxicity. The effective concentrations of these compounds a p p e a r e d to be much lower than the concentrations of N M D A antagonists. Subtypes of C C K receptors related to neuroprotective effects of CCK-8S were d e t e r m i n e d using competitive antagonists of C C K receptors. The binding studies have d e m o n s t r a t e d that the K i values of L-3647185 for the pancreas C C K g receptors and L-365260 TM for the brain C C K B receptors were 7 × 10 -11 M and 2 × 1 0 -9 M, respectively. T h e concentration (10 -s M) of the antagonists used in the present study was higher than the g i values of the drugs. Therefore, it is likely that the reduction of CCK-8S-induced neuroprotection by L365260 was due to the antagonism at C C K B receptors. C C K receptors are classified into two subclasses,

accumbens neurons were r e p o r t e d l y m e d i a t e d by C C K A receptors, but r e c e p t o r subtypes in o t h e r brain regions were not identified in these studies. In contrast to C C K A receptors, C C K B receptors have been considered to be central in type. H o w e v e r , functions of C C K B receptors in the CNS are not yet fully understood. The present results indicate that C C K - i n d u c e d protection against glutamate neurotoxicity is m e d i a t e d by C C K B receptors, since CCK-8NS, which acts selectively on C C K B receptors, p r o d u c e d n e u r o p r o t e c t i o n and a C C K B r e c e p t o r antagonist, L-365260, antagonized the effects of CCK. Therefore, the present study suggests the possibility that C C K B receptors play a role in the protection of cortical neurons against g l u t a m a t e - i n d u c e d d e g e n e r a t i o n in neurological disease state.

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The authors thank Dr. Chiyoko Inagaki (Department of Pharmacology, Kansai Medical University, Japan) for her generous advice on the techniques of maintaining cortical cultures. We thank Shionogi & Co., Ltd. (Japan) for kindly supplying ceruletide, L-364718, and L-365260.

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