Mechanisms of cholecystokinin-induced protection of cultured cortical neurons against N-methyl-d-aspartate receptor-mediated glutamate cytotoxicity

317

Brain Research, 592 (1992) 317-325 @ 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00 BRES 18037

Mechanisms of cholecystokinin-induced protection of cultured cortical neurons against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity Y u t a k a T a m u r a a, Y u k o S a t e

a,

A k i n o r i A k a i k e a a n d H i r o h i t o Shiomi

b

a 2nd and b 1st Departments of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama Unil'ersity, Fukuyama (Japan) (Accepted 7 April 1992)

Key words: Cerebral cortex; Cholecystokinin; Ceruletide; Glutamate; Neurotoxicity; Nitric oxide; N-MethyI-D-aspartate; Primary culture

The protective effects of cholecystokinin (CCK) against glutamate-induced cytotoxicity were examined using cultured neurons obtained from the rat cerebral cortex. Cell viability was significantly reduced when the cultures were briefly exposed to glutamate or N-methyI-D-aspartate (NMDA) and then incubatco with normal medium for 60 min. A fi0-min exposure to kainate also reduced cell viability. CCK protected cortical neurons against glutamate-, NMDA- and kainate-induced cytotoxicity. Glutamate- and NMDA-induced cytotoxicity was also reduced by N°'-nitro-Larginine, a nitric oxide (NO) synthase inhibitor. However, CCK did not prevent the cytotoxic effects of sodium nitroprusside (SNP) which spontaneously releases NO. Moreover, CCK did not affect NMDA-induced Ca2+ influx measured with rhod-2, a fluorescent Ca2+ indicator. Therefore, release of a NO-like factor from the cerebral cortex was assayed using the thoracic artery in vitro. When the artery was incubated with minced cerebral tissues, glutamate elicited marked relaxation. SNP also elicited relaxation of the smooth muscle. CCK inhibited glutamate-induced relaxation but did not affect that induced by SNP. These results indicate that CCK prevents NMDA receptor-mediated cytotoxicity without reducing the Ca2+ influx, it is suggested that CCK inhibits NO-formation triggered by NMDA receptor activation.

INTRODUCTION in the cerebral cortex, cholecystokinin (CCK) occurs predominantly in non-pyramidal bipolar cells 2J. The addition of depolarizing concentrations of K + or glutamate evokes CCK release "~4.Sulfated CCK oetapeptide (CCK-8S) is the major component of the endogenous CCK-related peptides in the brain including the cerebral cortex t4'3t. Since excitation is the predominant effect of iontophoretieally applied CCK.8S on cortical neurons, it has been suggested that CCK-8S acts as an excitatory transmitter or modulator in the cerebral cortex ~''~°. Moreover, we previously found that CCK-8S and other CCK-related peptides prevented glutamate cytotoxicity via CCK B receptors in cultured cortical neurons t, CCK may have a role in protecting cortical neurons against glutamate-induced cytotoxicity. Recent evidence has suggested that glutamate mediates the neurotoxicity observed in hypoxic.isehemic

brain injury 8,2"~.Moreover, brief glutamate-exposure induces delayed death in cultured neurons of the cerebral cortex 7'~, hippocampus 2s and cerebellum ~5. It has been postulated that the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is the predominant route of glutamate neurotoxicity since selective antagonists of the NMDA receptor prevent neuronal degeneration both in animal models of isehemia s''3 and in cultured neurons t°'2°, Glutamate neurotoxicity mediated by NMDA receptors involves Ca 2+ influx into cells via ligand-gated ion channels 24'2~. Recently, Dawson et ai. ~'~ have demonstrated using primary cortical cultures, that nitric oxide (NO) mediates the NMDA receptor-mediated neurotoxicity of glutamate, in their study, N'%nitro-L-arginine (identical to L-N~-nitro arginine), a NO synthase inhibitor 14'2s, prevented the neurotoxicity elicited by NMDA and related excitatory amino acids (EAAs). Moreover, hemoglobin, which complexes NO, prevented neurotoxic effects of both

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

318 NMDA and sodium nitroprusside (SNP), which spontaneously releases NO. NO, apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed in brain tissues ~'ta. Immunocytochemical studies have demonstratec" t.he p~-¢sence of NO synthase in select neuronal populations 01' the brain including the cerebral cortex 4. It has been proposed that NMDA receptor activation causes Ca 2+ influx, after which the cytosolic Ca 2+, working in conjunction with the regulatory protein calmodulin, turns on the synthesis of NO. NO diffuses to adjacent cells, resulting in the appropriate physiological responses and/or glutamate-related cell death. This evidence indicates that glutamate neurotoxicity includes several cellular responses; NMDA receptor activation, increase in cytosolic Ca 2+ concentration, activation of NO synthase by Ca2+-calmodulin complexes and synthesis of NO. Since CCK-related peptides prevent NMDA receptor-mediated glutamate neurotoxicity I, it is possible that CCK inhibits a step in the glutamate-induced responses mentioned above. To determine which response is suppressed by CCK, we used rat primary cortical cultures and demonstrate that CCK-gS and ceruletide, a CCK-related decapeptide 22, inhibit NO-formation without affecting Ca 2÷ influx via NMDA receptor-ion channel complexes. MATERIALS AND METHODS Cc# cub,a, Primary cultures obtained from the cerebral cortex of fetal rats (16-18 day gestation) were used for the experiments. "['he procedures have been described previously t, Briefly, single-ceils mechanically dissociated from the whole cerebral cortex of fetal rats (16-18 day gestation) were plated on plastic or glass coverslips which were placed in Falcon 60 mm dishes. Cultures were incubated in Eagle's minimal essential medium (MEM, Eagle's salt) supplemented with 10% heat-inactivated fetal bovine serum (!-9 days after plating) or 10% heat-inactivated horse serum (10-14 days after plating), glufamine (2 raM), glucose (total 11 raM), NaHCO 3 (24 raM), and HEPES (It) raM). Cultures were maintained at 37°C in a humidified 5% CO 2 atmosphere. After 8 days of plating, non-neuronal cells were removed by the addition of 10 -5 M cytosine arabinoside. Only mature (10-14 days in vitro)cultures were used in the study.

Measurement of neurmoxicity Neurotoxicity induced by EAAs was quantified by examining cultures under Hoffman modulation microscopy at ×400, Experiments were carried out in Eagle's solution at 37°C, After drug exposure, cell cultures were stained with !.5% Trypan blue for 10 rain at room temperature, fixed with isotonic formalin (oH 7.0, 2-4°0, then rinsed with physiological saline, Cells stained with the Trypan blue dye were regarded as non-viable. Over 200 cells were counted to determine the viability of the cell culture. In each experiment. 5-6 coverglasses were used to obtain means ± S.E.M. of the cell viability. Drug protection against EAA-induced neurotoxicity was calculated using the following equation: D-E Protectk)n (%) = ~ x I00

o.,°r.,,

IIg II olllf II1

/'N

JI

hol-plate stwrer

Fig. 1. Schematic diagram of the apparatus used for the simultaneous incubation of the aorta and minced brain tissues. Both inner and outer baths were made of glass. Solution in the inner bath was continuously stirred by a magnetic stirre~'.

in which D is the viability of the cell cultures treated with a drug and EAA, E is the viability after EAA treatment and C is the viability of non-treated cultures.

Measurement of cytosolic Ca 2 ÷ In order to assess cytosolic Ca 2+ concentration, cultures grown on coverglasses were loaded with rhod-2/AM (3.3/.~M, 30 min at 37°C). Experiments were performed at 37°C in HEPES-buffered Krebs solution (with low Mg 2+) containing (in raM): NaCI 116.4; NaHCO 3 26.2; KCI 5.4; MgSO4 0.01; HEPES 3.0; CaCI 2 1.8; and glucose I !.0 (pH 7.2). Rhod-2 fluorescence in the culture was measured using a fluorescence spectrophotometer (Hitachi, F-2000; excitation, 553 nm; emission, 576 nm according to the study of Minta et al."s). Drugs were added to the incubating medium which was continuously stirred on a magnetic stirrer. To estimate the total Ca"~* influx of each culture, ionomycin, a calcium ionophore, was added to the medium 10 min after NMDA. Measuremem of NO.like factor rek,ast,d [rom the cetebnd cortex Form,tion of , NO.like factor from the cerebral cortex was me,sured es described by Garthwalte et al. Ls with modifications. Male rats (200-300 g) were used in these experiments. After decapitation, the thoracic aorta and brain were quickly removed. The thoracic aorta was cleared of extraneous connective tissue and cut into rings, Endothelial cells were removed by rubbing the inner surface of the artery with moistened cotton wool. These preparations failed to relax in response to ACh (10 -~ M) but responded normally to phenylephrine (10-4 M). Both hemispheres of the cerebral cortex were dissected from the brain in fresh Krebs solution at 1-4°C. The tissues were cut into small pieces of about ! m m 3. Aortic rings were mounted using stainless steel wires under a tension of 0.5 g and were incubated with minced brain preparation in a 20 ml organ hath as shown in Fig. I, in some experiments, brain tissues were not added to the bath, The organ bath contained warmed (37°0, oxygenated (95% O,,, 5% CO 2) Krebs solution containing (in raM): NaCI 116.4; NaHCO3 26.2; KCI 5.4; NaH2PO 4 0.92; MgSO4 1.3; CaCI 2 2.5; and glucose, 11.0 (pH 7.2-7.4). After an iuitial equilibration period of 3 h (with several changes of the bath solution), tissues were contracted with phenyicphrine (10 -4 M). Isometric tension was recorded and drugs were added in 20 ~1 volumes. Drugs CCK-octapeptide sulfated form (CCK-8S, Peptide Institute), ceruletide (synthesized at Shionogi), ionomycin (Calbiochem), arid Rhod-2 acetoxymethyl ester (Rhod-2/AM. Dojindo Laboratories) were dissolved in distilled water and stored below -10°C. Acetylcholine chloride (Sigma), hemoglobin (Sigma), kainate (Nacalai Tesque), MK-801 (Research Biochemicals), monosodium glutamate (Nacalai Tesque), N'~-nitro-L-arginine (Sigma), N-methyl-o-aspar-

319

A

B

a

a

b

b

C

Fig. 2. Effects of CCK-8S on neurotoxicity induced by NMDA (A) and kainate (B). Culture fields were photographed after Trypan blue staining followed by formalin fixation. Aa and Ba indicate the non-treated cells (control). Ab were cells treated with NMDA (0.5 raM) for 10 rain in Mg2+-free medium and further incubated with Mg2+-free medium without NMDA. Bb were cells treated with kainate (0,5 raM) for 60 rain in Mg2+-containing medium. Ac and Bc represent cells treated with CCK-8S (10 -7 M) plus NMDA and cells treated with CCK-8S (10 -7 M) plus kainate, respectively. Calibration bar = 50/~m.

320 A

TABLE i

Cytotoxicity induced by exc'tatory amino acids (500 p M ) in the pres¢,~¢ arm absence o f Mg " +

Time Mg z + Viability (%)

(min) (raM) None (control)

Glutamate

Protectlon(%) 50

10O

50

100

50

1O0

CCK

Data are expressed as means±S.E.M. ( n = 5).

Drug

O

CLT

Mg" + Viability (raM) (%)

1.6

91.2 + 0.9

Glutamate Glutamate

10 a 60 b

1.6 1.6

42.8 + 1.3 * * 0 39.4+1.5 ** 0

38.7+0.8 ** 36.4 +0.9 **

NMDA NMDA

10 a 60 b

1.6 1.6

89.7 "l-1.8 74.5+3.1

41.6+0.3 ** 40.5 + 3.3 **

Kainate Kainate

10 a 60 t,

1.6 1.6

89.2+0.3 44.7 + 1.6 * *

0 0

MK-8Ol

B

O

NMDA

CCK

** P < 0.01 (compared with control). a Cultures were treated with amino acids for 10 rain followed by an incubation with medium, which did not contain excitatory amino acids, for 60 rain. b Cultures were treated with amino acids for 60 rain.

tale (Sigma), t.-phenylephrine hydrochloride (Sigma), and sodium nitrop,usside (Wake) were dissolved in Eagle's or Krebs solution immediately prior to the experiments. Statistics Data were expressed as mean ± S.E.M. The statistical significance of the data was determined by Dunnett's two-tailed test.

RESULTS

Cytotoxicity induced by EAA~ Table I summarizes the cytotoxic effects of 0..5 mM EAAs. In our previous study t, a marked reduction of the cell viability was induced by exposing the cultures to glutamate (0.5-1 mM) for 10 rain followed by incubation in glutamate.free medium for more than 60 min. Therefore, we compared the cytototoxic effects of glutamate, NMDA and kainate using two exposure times: (a) a 10-rain exposure to EAA followed by 60 min incubation in EAA-free medium and (b) a 60-rain exposure to EAA. Both I0- and 60-min exposures to glutamate induced a significant reduction in the cell viability. Glutamate-induced cytotoxicity was observed in the presence and absence of Mg 2+. By contrast, NMDA added to Mg' +-containing medium did not induce significant cytotoxicity. In Mg:+-free medium, both I0- and 60-rain exposure to NMDA induced significant reduction of the celt viability. In contrast to glutamate and NMDA, a 10-rain exposure to kainate did not induce significant cytotoxicity. Kainate-induced cytotoxicity was observed only after 60 rain.

CCK-induced protection against EAA neurotoxicity Since the previous study t demonstrated that CCKrelated peptides including CCK-8S at the concentra-

CLT MK-801

I

C

0

Kainate

I. . . . . . . . . . . . . . . . CCK

MK-801

~ I

Fig, 3. Effects of CCK-related peptides and MK-801 on cytotoxicity induced by glutamate (A), NMDA (B) and kainate (C). Each column and bar represent mean and S,E.M. of data (n = 5), respectively. Protection (%) in the abscissa was calculated using the equation described under Materials and Methods. Cultures were treated with glutamate (0.5 mM) or NMDA (0.5 mM) for I0 rain then incubated with EAA-free medium for 60 rain, or treated with kainate (0.5 raM) for 60 rain, Drugs were added to both the EAA.containing and EAA-free media. Mg'+-free solution was used for NMDA studies, Respective drugs and concentrations were CCK-SS (CCK), 10 -7 M;

ceruletide (CLT), 10-7 M; and MK-801, 10-'~ M.

tion of I0 -~ M produced maximum protection against glutamate-induced cytotoxicity, we examined the effects of CCK-8S at I0 -v M on cytotoxicity induced by NMDA or kainate. Fig. 2 shows an example of CCK8S-induced protection against EAAs. A 10-min exposure of the cells t o NMDA (I mM, added in the Mg~+-free medium), followed by a 60-min incubation, markedly increased the number of cells stained by Trypan blue (Fig. 2Ab). A 60-min exposure to kainate (I raM, added in the Mg'÷-containing medium) also increased the number of stained cells (Fig. 2Bb). Concomitant addition of CCK-8S (I0 -~ M) with NMDA or kainate reduced the number of cells stained by Trypan blue (Fig. 2Ac and Be). Fig. 3 summarizes the protective effects of CCK-related peptides and MK-801 against glutamate-, NMDAand kainate-induced cytotoxicity. CCK-8S (I0 -~ M) and ceruletide (I0 -~ M) significantly reduced the cytotoxic effects of all the EAAs tested. By contrast, MK801 (10 -5 M), a selective blocker of NMDA receptors9'33, did not affect kainate-induced cytotoxicity

321 100

though the drug prevented glutamate- and NMDA-induccd ~'totoxicity. Participation of NO in NMDA receptor-mediated neurotoxicity Recently, Dawson et al. ~3 reported that nitric oxide (NO) mediates NMDA receptor-mediated glutamate neurotoxicity in primary cortical cultures. Therefore, to determine whether or not CCK prevents the neurotoxic effects of NO, cells were treated with SNP which spontaneously releases NO 32. First, we examined the effect of N°'-nitro-L-arginine, a NO synthase inhibitor t5'26, on glutamate-induced cytotoxicity to determine whether or not NO mediated glutamate neurotoxicity in the cortical cultures used in this study (Table II). N°'-nitro-L-arginine (0.3 mM) significantly reduced glutamate- and NMDA-induced cytotoxicity but did not affect that induced by kainate. This indicates that NO mediates the NMDA receptormediated eytotoxi¢ity of glutamate in the cortical cultures maintained in our laboratory. A brief exposure of the cells to SNP (0.5 raM) followed by 60 rain incubation in SNP-free medium markedly reduced viability of cultures (Fig. 4). Hemoglobin (10 -5 M), which binds NO t3' prevented the cytotoxicity induced by SNP. If CCK receptor stimulation facilitated the activities of the intracellular systems to remove NO itself, or radicals synthesized by

0 C 0 O

"~ 5O E 0

._L

._L_ _a_

0..

o SNP .

. . . Hb CCK CLT MK-801 Fig. 4. Cytotoxicity induced by brief exposure to sodium nitroprusside (SNP, 5 × 10 - 4 M). Ordinate represents viability of cultures expressed as a percent of control (non-treated cultures). Cultures were treated with SNP for 10 min then incubated with normal medium for 60 rain. Drugs were added to SNP-containing solution. Respective drugs and concentrations were hemoglobin (Hb), 10 -5 M; CCK-8S (CCK), 10 -7 M; ceruletide (CLT), 10 -7 M; and MK-801, 10 -5 M.

NO, CCK or ceruletide should prevent SNP-induced cytotoxicity. However, CCK-8S ( 1 0 - 7 M ) and ceruletide (10 -7) failed to protect cells against SNP-induced cytotoxicity. MK-801 (10 -3 M) was also ineffective in protecting cells against SNP cytotoxicity.

TABLE II

Effects of N'°.L-nitro.argitdne (N.Arg) tm neurot~xcicity induced by etcitatory amino acids Data are expressed as means + S.E.M. ( n m 5).

Treatment

Viability

(%)

Protection

(%)

None (control)

88.7 + 1.3

-

Glutamate (l raM) a Glutamate (1 raM) + N-A~g (0.3 mM) b

34,5 +2.5

-

NMDA (1 raM) c NMDA (1 mM) + N-Arg (0.3 raM)

38.2:1:1.4

Kainate (1 mM) a Kainate (1 mM) + N-Arg (0.3 raM)

36.4+0.8

76.3 + 0.8 * *

77.3 + 0.9 * *

44.6+0.6

77.1 5:1A 77.4 + 1.8

15.7+ 1.1

** P < 0.01 (compared with the respective excitatory amino acid alone). a Cultures were treated with glutamate for 10 rain followed by an incubation with glutamate-free medium for 60 rain. b N-Arg was added to the medium containing excitatory amino acids. Cultures were treated with NMDA for 10 rain followed by an incubation with NMDA-free medium for 60 rain. MgZ+-free medium was used. a Cultures were treated with kainate for 60 rain.

Effects of CCK on NMDA.induced Ca 2 + influx The effects of CCK on NMDA-induced Ca2÷-influx were examined to determine whether or not CCK affected the agonist-induced activation of NMDA receptor-gated ion channels. The cytosolic Ca 2+ concentration was measured using rhod-2 25. NMDA (0.5 raM) added to the medium containing low concentration (0.01 raM) of Mg 2+ evoked an immediate increase in Ca2+-fluorescence (Fig. 5Aa and B). Moreover, a persistent increase in the cytosolic Ca 2+ was observed in the presence of NMDA. Ionomycin (5 × 10 -° M), a calcium ionophore, was added to the medium 10 rain after the addition of NMDA to estimate the total Ca 2+ influx. Calcium influx induced by NMDA war determined as the percentage of the ratio of an apparent NMDA-induced influx against an apparent ionomycin influx as shown in Fig. 5B. CCK-8$ (10 -7 M), ceruletide ( 1 0 - 7 M) and MK-801 (10 -5 M) were added to the medium 2 rain before NMDA. Neiti.zr CCK nor ceruletide affected NMDAinduced Ca 2+ influx. By contrast, MK-801 (10 -5 M) significantly reduced NMDA-induced Ca 2+ influx.

322

Effects of CCK on the formation of NO-like factor

mt~.t) in the presence of the brain tissues caused a p~[sistc,,t ,claxatio,, corresponding in magnitude, to approximately 100% of the phenylephrine-induced contracture. Glutamate at 0.1-1 mM was ineffective in eliciting relaxation of the aorta. CCK-8S (10 -7 M) added following glutamate application completely abolished glutamate-induced relaxation (n = 3). Ceruletide (10 -7 M) added following glutamate-induced relaxation also abolished glutamate-induced relaxation with a similar potency to that of CCK-8S (n = 3). SNP (5 x 10 -4 M) added following CCK-8S or ceruletide elicited a prompt relaxation, the magnitude of which was approximately 100% of the CCK-8S- or ceruletiderestored contraction (n -- 6).

Phcnylcphrinc at concentrations of 10-~-10 -4 M dose-dependently induced contraction of the aortic ring, from which the endothelium was removed (Fig. 6A). The aorta contracted by phenylephrine was not relaxed by ACh (up to 10 -3 M), but was promptly relaxed by the subsequent addition of SNP (5 x 10 -4 M). CCK-8S (10 -7) itself did not affect the resting tension or phenylephrine-induced contraction of the aorta (Fig. 6B and C). In the following experiments, the aorta was precontracted with phenylephrine (10 -4 M) which was present throughout the rest of the experiment. When the aorta was maintained in the absence of the cerebral tissues, glutamate (10 mM) did not elicit a change of ~ension although subsequent addition of SNP (5 × 10 -4 M) elicited prompt relaxation (Fig. 7A). CCK (10 -7 M) added following SNP did not inhibit SNP-induced relaxation (n = 4). NO-like factor released from the cerebral cortex was assessed by incubating the aorta with minced brain tissues. As shown in Fig. 7B, adding glutamate (10

A

Control

a

b

520 - -

540 •

S

450 , i 080 ,

280

ILl01

J

"°"

DISCUSSION The present study demonstrates that very low concentrations (10 -7 M) of CCK-8S and ceruletide potently inhibit glutamate neurotoxicity mediated not only by NMDA receptors but also by kainate receptors. Both 10-rain and 60-min exposures to glutamate caused

CCK-8S

10-TM

NK-80I qt~O

/f

420 -

G20 800

320

"

/

6GO b30

i,:i: 000

300

900

.

500

. . . . . . . . .

000

94)0

600

(,o~)

B

lo-SH

C -

N

j ~

,r,f

3()0

BOO

C

9OO

(auo)

(-oe)

70,

TOO

1" [ ' "

,°o

~

,°'

"

L

"

b

~

4o,

i 200

I

~ m

i|0

EO0

120

(see)

(see)

(s,e)

0 NHDA SOQ/AN CCK-BS

CLT

HK-801

Fig. 5. Effects of CCK-related peptidcs and MK-801 on.NMDA-indnccd changes in intraceUular Ca2~ concentrations. A: examples of intraceqular Ca~÷ changes induced by N M D A (0,5 raM) in the absence (a) and presence of CCK (b) and MK-801 (c); C~K and MK-801 were added to the medium 2 rain before NMDA application, Arrows N and ! in the recording traces respectively indicate the addition of NMDA (0,5 raM) and ionomycin~4 (5 pM), B: the method to estimate total Ca 2+ influx induced by N M D A (a) and ionomycin (b), C: summary of the effects

of CCK (10-? M), ceruletide (CLT, 10-7 M) and MK-80I (10-s M) on NMDA-inducedCa2+ influx. The ordinate indicates the percentage of the ratio of NMDA-inducedinflux(Ba) against ionomycin-inducedinflux(Bb).

323

A Phe -14-13-12.-11-10-9-8

ACh SNP -7

- 6 -S -4

-3-3,3

B CCK -7

0.5g

10 mln

C

Phe -4

CCK -9

..qr._v . ~ '-ww"! "~'*,q~r~

-8

-7

SNP - 6 -3.3

P WI~ 11

Fig. 6. Effects of CCK-SS on phenylephrine-induced contraction of the thoracic aorta, The aorta was incubated in the absence of brain tissue, A: dose-dependent contraction induced by phenylephrine, B: effects of CCK-SS on the resting tension. C: effects of CCK-8S on phenylephrine-induced contraction. Respective drugs and concentrations were phenylephrine (Phe), 1 0 - 1 4 10-4 M; acetylcholine (ACh), 10='~ M; sodium nitmprusside (SNP), 5 x l 0 -4 M; and CCK-8S (CCK), 10- ~ M. Numbers above the arrows indicate logarithms of molar concentrations of the dru~s.

similar reductions in the cell viability. This indicates that 10 t i n is long enough to trigger glutamate-induced delayed death. N M D A was cytotoxic in a manner similar to that of glutamate, except that the absence of Mg 2+ in the incubation medium was required. NMDA-induced neurotoxicity was not observed when the incubating medium included normal concentrations (1.6 raM) of Mg 2+. These results concerning N M D A neurotoxieity are consistent with electrophysiological evidence indicating that N M D A re~,~ptor-gated ion channels are blocked by Mg 2+ at the resting m e t brahe potential tl'2?'2s, in contrast to glutamate or NMDA, a ] 0 - t i n exposure to kainate was not cytotoxic whereas a 60-min exposure in the presence of Mg 2+ induced pronounced cytotoxicity. Probably, the relatively long-term stimulation of kainate receptors is necessary to induce cell death unless NMDA receptors are not stimulated. These results indicate that NMDA receptor is a major cause of the delayed death induced by brief exposure to glutamate. This assumption is also

supported by the previous studies in our laboratory' and others 1°';°, demonstrating that delayed ceil death following brief exposure to glutamate was prevented by NMDA ~'eceptor antagonists. It is likely that glutamate first stimulates non-NMDA receptors such as kainate receptors, which are not regulated by Mg 2+, to depolarize cells, then N M D A receptors are activated following the reduction of Mg 2÷ blockade by depolarization. Therefore, the present study was focused upon N M D A receptor-mediated neurotoxicity, although CCK-re]ated peptides prevented both the N M D A receptorand kainate receptor-mediated neurotoxicity. The present study also showed that N=-nitro-L arginine, a selective inhibitor of NO synthase1s'26, reduced glutamate- and NMDA-induced cytotoyJcity but did not affect that induced by kainate. Moreover, a brief exposure to SNP, which spontaneously releases NO 13,32,induced delayed neurotoxicity. Cytotoxicity of SNP was prevented by hemoglobin, which traps NO released in the medium. These results are consistent with a study by Dawson et al. j3, who demonstrated that NO mediates glutamate neurotoxicity in cultured cortical neurons. However, the present study showed that CCK-SS and ceruletide did not prevent neurotoxicity

A

Without brain Phe

B

Glu

SNP CCK

0,00

With brain Phe

Gtu

CCK

SNP

Fig. 7. Effects of CCK-8S on glutamate-induced relaxation in the presence of cerebral tissues. The thoracic aorta was incubated either in the absence (A) or in the presence (B) of cerebral tissues. Respective drugs and concentrations were phenylephrine (Phe), 10-4 M; glutamate (Glu), 10-2 M; sodium nitroprusside (SNP), 5 × 10-4 M; and CCK-8S (CCK), 10-~ M.

324 induced by SNP. Therefore, it is unlikely that CCK receptor stimulation facilitates the activity of the intracellular systems which remove NO or radicals synthesized by NO. Although there is no evidence to indicate that CCK-related peptides directly attenuate ligand binding of NMDA receptors, CCK receptors may indirectly interact with NMDA receptors via second messenger systems. If this is so, Ca2+-influx induced by NMDA receptor stimulation must be affected by CCK-related peptides. In order to examine this possibility, we assessed the cytosolic Ca 2+ concentration using rhod-2. The high concentration (0.5 raM) of NMDA could evoke a large Ca 2+ influx. Therefore, we used rhod-2 25 as the fluorescent indicator for cytosolic calcium since it gives better resolution for high [Cae+]i than that of fura-2 due to its large effective K d value for Ca 2+. Elevation of cytosolic Ca 2+ concentration following exposure to NMDA is thought to be due to the activation of ion channels directly gated by NMDA receptors, since MK-801, a selective blocker of NMDA receptor-gated channels 't.-~s, significantly reduced NMDA-induced Ca '+ influx. In contrast to MK-801, CCK-8S and ceruletide did not affect NMDA-indueed Ca 2÷ influx. This indicates that CCK receptor stimula. tion does not inhibit the itgand-induced activation of NMDA receptor-ion channel complex. These results led us to assume that the neuroprotee. tire effects of CCK-related peptides may be caused by its inhibitory action on the intracellular response linked with NO-formation, but not by the reduction of Ca: + influx or NO cytotoxicity. To confirm this speculation, NO released from the cerebral cortex was assessed using the isolated aorta as a biological sensor of NO. Garthwaite et al. 's demonstrated that NMDA caused a relaxation of the aorta contracted with phenylephrine when the aorta was incubated with suspensions of single cells dissociated from the 8-9-day-old rat cerebella, in the present study, we used minced brain tissues dissected from adult rats to avoid enzyme treatment of the brain tissues. Glutamate had no effect on muscle tension when applied in the absence of the brain tissues. When the cerebral tissues were included in the bathing medium, however, glutamate elicited a marked relaxation. We used glutamate, but not NMDA, to avoid modifying incubating medium, since removal of Mg 2÷ was required to observe the NMDA-induced action 27"2s'35. CCK-related peptides abolished glutamate-induced relaxation in the presence of brain tissues. Since CCK-SS did not induce any effects on the tension of the aorta under normal conditions, it is likely that the CCK-induced blockade of glutamate-indtJced relaxation is due to a direct action upon the

CCK

Ca1, Na*

1

~ C Calmodulin

, )

Ca~*'Calm°dulinl1

NOSxnthase

1

NO (nitric oxide)

1 Cell Death Fig.8. Schematicrepresentationof the presumedmechanismsof the protectiveeffect of CCK-relatedpeptidesagainst NMDAreceptormediatedglutamatecyiotoxicity,

brain tissues, but not upon the smooth muscle. These results indicate that CCK-related peptides reduce glutamate-incited NO-formation in the cerebral cortex. Fig. 8 summarizes the mechanisms of CCK-inducod protection against NMDA receptor-mediated glutamate neurotoxicity in the cerebral cortical cultures according the scheme by Hoffman 2~. Activation of NMDA receptor-gated ion channels by glutamate causes elevation of cytosolic Ca :+ concentration, Since NO synthase is a calmodulin-requiring enzyme s, cytosolic Ca 2+ has a crucial role in NO-formation. Ca '+, working in conjunction with the regulatory protein calmodulin, activates NO synthase and facilitates formation of NO, which in turn diffuses to adjacent cells and mediates glutamate neurotoxicity ~6,:,t. CCK receptor stimulation presumably causes suppression of either a step in the Ca2+.calmodulin complex or NO synthase activation via second messenger systems, though the precise mechanisms concerning the intracellular mechanisms linked with C C K , receptors are yet to be determined. Thus, CCK appears to reduce cytotoxic effects of glutamate without inhibiting its excitatory effects. This neuroprotective effect of CCK is the first finding to suggest that certain neurotransmitters or modulators released in the brain protect neurons against the cytotoxic effects of glutamate. It has been postulated that the cytotoxic effects of EAAs, including glutamate, have a crucial role in the pathogenesis of the neuronal

325 degeneration which is associated with several neurological disease states 7"2°, However, it is also widely accepted that EAAs play a dominant role in excitatory transmission and long-term potentiation in the central n e r v o u s s y s t e m t2'tg. This suggests that neurons are continually exposed to both the excitatory and cytotoxic effects of glutamate. Therefore, it is possible that new roprotective substances such as CCK have a physiological role in avoiding rapid cortical neuronal degeneration. We therefore postulate that CCK is a putative neuroprotective factor against glutamate cytotoxicity in the cerebral cortex. Acknowledgements. The authors thank Dr. Keisuke Hattori (Department of Pharmacology, Shimane Medical University, Japan) for his generous technical advice. We thank Shionogi & Co., Ltd. (Japan) for kindly supplying ceruletide. REFERENCES 1 Akaike, A., Tamura, Y., Sate, Y., Ozaki, K., Matsuoka, R., Miura, S. and Yoshinaga, T., Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity, Brain Res., 557 (1991) 303-307. 2 Barden. N,, Merand, Y,, Rouleau, D., Moore, S,, Dockray, GJ. and Dupont, A., Regional distribution of somatostatin and cholecystokinin-like immunoreactivities in rat and bovine brain, Pep. tides, 2 (1981) 299-302. 3 Beinfeld, M.C., Meyer, D.K., Eskay, R.L., Jensen, R.T. and Brownstein, M.J., The distribution of cholecystokinin in the c~ntral nervous system of the rat as determined by radioimmunoassay, Brain Res,, 212 (1981) 51-57, 4 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770, 5 Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulln.requiring enzyme, Proc. Natl. Acad. Sci. USA, 87 (1990) 682-685. 6 Chiyodo, L.A., Freeman, A.S. and Bunney, B.S., Electrophysiological studies on the specificity of the ¢holecystokinin antagonist proglumide, Brain Res,, 410 (1987) 205-211, 7 Choi, D.W., Ionic dependence of glutamate neurotoxicity, ,/, Neurosci., 7 (1987) 369-379, 8 Choi, D.W., Calcium.mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, Trends Neurosci., 11 (1988) 465-469. 9 Choi, D.W., Maulucci-Gedde, M. and Kriegstein, A.R,, Glutamate neurotoxicity in cortical cell culture, J. Neurosci., 7 (1987) 357-368. 10 Choi, D.W., Viseskul, V., Amirthanayagam, M. and Monyer, H., Aspartate neurotoxicity on cultured cortical neurons, .I. Neurosci. Res., 23 (1989) 116-121. 11 Collingridge, G.L. and Lester, R.AJ., Excitatory amino acid receptors in the vertebrate central nervous system, Pharmacol. Rev., 40 (1989) 143-210. 12 Collingridge, G.L. and Singer, W., Excitatory amino acid receptors and synaptic plasticity, Trends Pharmacol. ScL, 11 (1990) 29U-296. 13 Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S, and Snyder, S.H., Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures, Proc. Natl. Acad. Set. USA, 88 (1991) 6368-6371. 14 Dorkray, GJ., Cholecystokinin in rat cerebral cortex: identification, purification and characterization by immunochemical methods, Brain Res., 188 (1980) 155-165.

15 Dwyer, M.A., Bredt, D.S. and Snyder. S.H., Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo, Biochem. Biophys. Res. Commun., 176 (1991) i136-1141. 16 Favaron, M., Manev, H., Siman, R., Bertolino, M., Szekely, A.M., DeErausquin, G., Guidoni, A. and Costa, E., Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death, Prec. Natl, Aead. Sci. USA, 87 (1990) 1983-1987. 17 Garthwaite, J., Glutamate, nitric oxide and cell-cell signaling in the nervous system, Trends Neurosci., 14 (1991) 60-67. 18 Garthwaite, J., Charles, S.L. and Che=s-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests as a messenger in the brain, Nature, 336 (1988) 385 -388. 19 Gustafsson, B. and Wigstr6m, H., Physiological mechanisms underlying long-term potentiation, Trends Neurosci., 11 (1988) 156162. 20 Hartley, D.M. and Choi, D.W., Delayed rescue of N-methyi-Daspartate receptor-mediated neuronal injury in cortical culture, 3. Pharmacol. Exp. Ther., 250 (1989) 752-758. 21 Hoffman, M., A new role for gases: neurotransmission, Science, 252 (1991) 1788. 22 Ibii, N., lkeda, M. Takahara, Y. Eigyo, M. Akiyoshi, T. and Matsushita, A., Inhibitory effect of ceruletide on haloperidol-induced catalepsy in rats, Peptides, 10 (1989) 779-783. 23 Meldrum, B. and Garthwaite, J., Excitatory amino acid neurotoxicity and neurodegenerative disease, Trends Pharmacol. ScL, i! (1990) 379-387. 24 Michaels, R.L. and Rothman, S.M., Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations, J. Neurosci., 10 (1990) 283-292. 25 Minta, A., Kao, J.P.Y. and Tsien, R.Y., Fluorescent indicators for cytosolic calcium based on rhodamine and fluoreseein chromophores, J. Biol. Chem., 264 (1989) 8171-8178. 26 Moore, P.K., AI-Swayeh, O.A., Chong, N.W S., Evans, R.A. and Gibson, A., L-NG-nitro arginine (L-NOAKG), a novel, Larginine-reversible inhibitor of endothe~ium-dependent vasodilafinn in vitro, Br. J. Pharmacol., 99 (1990) 408-414. 27 Moriyoshi, K., Masu, M., Ishii, T., Shigemoto, R., Mizuno, R. and Nakanishi, S., Molecular cloning and characterization of the war NMDA receptor, Nature, 354 (1991) 31-37. 28 Nowak, L,, Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates glutamate-activated channels in mouse central neurons, Nasu~, 307 (1984) 462-465. 29 Ogura, A., Miyamoto, M. and Kudo, Y., Neuronal death in vitro'. parallelism between survivability of hippocampal neurones and sustained elevation of cytosolic Ca 2+ after exposure to glutamate receptor agonist, Exp, Brain Res., 73 (1988) 447-458, 30 Phillis, J.W. and Ktrkpatrick, J.R,, The actions of motilin, lutenizing hormone releasing hormone, cholecystokinin, somatostatin, vasoactive intestinal peptide, and other peptides on rat cerebral cortical neurons, Can. J. Physiol. Pharmacol., 58 (1980) 612-623. 31 Rehfeld, J.F., Immunological studies on cholecystokinin II. Dis. tribution and molecular heterogeneity in the central nervous system and small intestine of man and hog, J. Biol. Chem., 253 (1978) 4022-4030. 32 Waldman, S,A, and Murad, F., Cyclic GMP synthesis and formation, Pharmacol. Rev., 39 (1987) 163-196. 33 Wong, E.H.F., Kemp, J.A,, Priestley, T., Knight, A.R., Woodruff, G,N. and Iversen, L.L., The anticonvulsant MK-801 is a potent N.methyI-D-aspartate antagonist, Prec. Natl. Acad. Sci. USA, 83 (1986) 7104-7108. 34 Yaksh, T.L., Fun)i, T., Kanawati, I.S. and Go, V.L.W., Release of cholecystokinin from rat cerebral cortex in vivo: role of GABA and glutamate receptor systems, Brain Res., 406 (1987) 207-214. 35 Young, A.B. and Fagg, G.E., Excitatory amino acid receptors in the brain: membrane binding and receptor autoradiographic approaches, Trends Pharmacol. 3ci., 11 (1990) 126-133.