해마절편에서 저 산소증에 의한 5-hydroxytryptamine 유리변동과 N-methyl-D-aspartate 수용체와의 관련성

Abstract

[한글]

뇌기능은 뇌혈류가 18ml/100g/min 이하로 감소되면 허혈성 장애를 나타내며 혈류가 완전 차단될 경우 수분이내에 불가역적인 뇌의 손상을 일으키게 된다. 최근 허혈의 치료로는 신경세포자체의 취약성을 개선시켜 주는 방법이 시도되고 있으며, 이러한 관점에서 흥

분성 아미노산 신경전달물질의 독작용에 대한 관심이 증대되고 있다. 허혈시 glutamate는 신경말단에서 유리가 증가되고 재흡착 장애로 신경말단내에서 증가한다. 이때의 glutamate는 접합후 수용체, 특히 N-methyl-D-aspartate(NMDA)수용체와 결합하여 세포내로 칼슘을 유입시킴으로서 지연성 신경세포 독작용을 유발한다. 또한, 허혈성 뇌손상은 아민계 신경전달물질과도 밀접한 연관이 있으며 허혈시 나타나는 이들 신경전달물질의 변동은 신경세포손상의 간접적인 지표로 사용될 수 있다. 그러나 이에 관하여는 아직 일관된 견해

가 없으며 특히 허혈과 관련있는 아미노산계 및 아민계 신경전달물질 상호간의 연관성에 대하여는 연구가 부족한 실정이다. 따라서 이번 실험에서는 저산소 상태에 민감한 흰쥐 해마의 절편을 이용하여 NMDA 또는 이의 봉쇄제 투여 전후에 따른 (3)**H -5-hydroxytryptamine((3)**H -5-HT) 유리의 변동양상을 검색하여 저산소 상태에서 흥분성 신경전달물질의 역할을 규명하고 5-HT성 신경과 glutamate성 신경의 상호작용을 밝히고자 하였다. 횐쥐의 뇌를 적출하여 얻은 해마절편을 30분간 영양액에 방치하여 안정시킨후 0.laM의 (3)**H -5-HT(74μCi)가 포함된 영양액에서 2D분간 (3)**H -5-HT를 흡착시킨 다음 세척하였다. 실험군은 대조군, 저산소 노출군(95% N^^2 /5% CO^^2 포화 영양액에 10분간또는 20분간

노출), NMDA(1 mM)투여군, 저산소 노출 및 NMDA 동시 투여군 및 NMDA봉쇄제(2-amino-5-phosphonovaleric acid, APV, 30μM) 투여군으로 하였으며 APV는 저산소 노출이나 NMDA투여 10분전부터 이들_ 처치동안 계속 투여하였다. (3)**H -5-HT 유리는 매 10분간격으로 140분간 반복하여 영양액을 갈아주며 측정하였다. (3)**H -5-HT 유리양은 영양액 내로 유리된 (3)**H -5-HT양과 조직에 남아있는 양을 liquid scintillation counter로 측정하여 총 방사능량에 대한 각각의 백분율로 표현하였다. 이번 실험에서 얻어진 결과는 다음과 같다.

1. 정상 영양액 내에서 (3)**H -5-HT는 해마절편으로부터 자발적으로 유리되었으며 그 유리양은 50분 후부터는 비교적 일정하였다.

2. 저산소 영양액에 해마절편을 노출하였을 때 (3)**H -5-HT 유리는 현저히 감소되었으며 이는 정상 영양액으로 환원하였을 경우 10분간의 저산소 노출군에서는 지속적인 (3)**H -5-HT 유리감소를 나타내었으나 20분간의 저산소 노출군은 정상 영양액으로 환원할 경우 대조군보다 높은 (3)**H -5-HT의 반동성 유리증가를 나타내었다

3. 정상 영양액 내에 NMDA를 투여하면 해마절편은 대조군에 비하여 의의있는 (3)**H -5-HT 유리증가를 나타내고 이러한 유리증가는 APV에 의하여 용량 의존적으로 봉쇄되었다.

4. 해마절편에 저산소 영양액 노출과 아울러 NMDA를 동시에 투여하였을 때 저산소 영양액 노출동안 나타나는 (3)**H -5-또T 유리감소가 봉쇄되었으며 반동성 증가는 더욱 심하였다.

5. APV 투여는 저산소 영양액 노출후 정상 영양액으로 환원하였을 때 나타나는 반동성 증가를 완전히 봉쇄하였다.

이상의 성적으로 보아 저산소 영양액 노출은 (3)**H -5-HT 유리감소를 나타내고 정상 영양액으로 환원하면 20분간 저산소에 노출된 경우에 반동성 (3)**H -5-HT 유리증가를 일으키며 이러한 반동성증가에 NMOA 수용체 활성이 관여할 것으로 생각된다.

Involvement of N-methyl-D-aspartate receptor in the release of 5-hydroxytryptamine

after hypoxia from rat hippocampal slices

Sung-Hee Hwang

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professor Young Soo Ahn)

Hypoxic insult increases the level of extracellular glutamate, which leads to the

influx of toxic Ca**++ through the activation of N-methyl-D-aspartate(NMDA)

receptors. The neuroprotective action of NMDA antagonist against hypoxic insult has

been demonstrated in vitro. It has been demonstrated that the concentration of

5-hydroxytryptamine(5-HT) also increased after ischemia in rat hippocampus.

However, there is paucity of studies concerning the functional relationships

between the spontaneous release of 5-HT and NMDA receptor activity during hypoxia

in vitro. Therefore, the present study was aimed to investigate whether hypoxia

and/or NMDA was able to stimulate the release of 5-HT from the hypoxia-sensitive

rat hippocampal slices.

The hippocampus was obtained from the rat brain and sliced 400μm thickness with

manual chopper. After 30 min's preincubation in the normal buffer, the slices were

incubated for 20 min in a buffer containing (3)**H -5-HT (0.1μM, 74μCi) for

uptake, and washed. To measure the release of (3)**H -5-HT into the buffer, the

incubation medium was drained off and refilled every ten minutes through a sequence

of 14 tubes. Administration of NMDA or induction of hypoxia(gassing it with95%

N7/5% CO,) was done in the 6th and 7th tube, and APV was added 10 minutes prior to

these manipulations. The radioactivities in each buffer and the tissue were counted

using liquid scintillation counter and the results were expressed as a percentage

of the total radioactivity.

When slices were exposed to hypoxia for 20min, (3)**H -5-HT release was markedly

decreased and are bound release of (3)**H -5-HT was observed on the post-hypoxic

period. NMDA(1 mM) increased (3)**H -5-HT release in the control group. NMDA also

increased rebound release of (3)**H -5-HT in post-hypoxic period and prevented

hypoxia-induced decrease of (3)**H -5-HT release. When 2-amino-5-phosphonovaleric

acid(APV, 307M or 607M ) were added to the incubation media, NMDA-induced increase

of (3)**H -5-HT release was blocked dose-dependently. The rebound release of (3)**H

-5-HT during post-hypoxic period was also blocked by APV.

These results suggest that the spontaneous release of (3)**H -5-HT decreases

during hypoxic period, but 20min hypoxic exposure causes rebound increase of (3)**H

-5-HT release during post-hypoxic period which is mediated by the increased

activity of the NMDA receptor.

[영문]

Hypoxic insult increases the level of extracellular glutamate, which leads to the influx of toxic Ca**++ through the activation of N-methyl-D-aspartate(NMDA) receptors. The neuroprotective action of NMDA antagonist against hypoxic insult has been demonstrated in vitro. It has been demonstrated that the concentration of 5-hydroxytryptamine(5-HT) also increased after ischemia in rat hippocampus.

However, there is paucity of studies concerning the functional relationships between the spontaneous release of 5-HT and NMDA receptor activity during hypoxia in vitro. Therefore, the present study was aimed to investigate whether hypoxia and/or NMDA was able to stimulate the release of 5-HT from the hypoxia-sensitive

rat hippocampal slices.

The hippocampus was obtained from the rat brain and sliced 400μm thickness with manual chopper. After 30 min's preincubation in the normal buffer, the slices were incubated for 20 min in a buffer containing (3)**H -5-HT (0.1μM, 74μCi) for

uptake, and washed. To measure the release of (3)**H -5-HT into the buffer, the incubation medium was drained off and refilled every ten minutes through a sequence of 14 tubes. Administration of NMDA or induction of hypoxia(gassing it with95% N7/5% CO,) was done in the 6th and 7th tube, and APV was added 10 minutes prior to these manipulations. The radioactivities in each buffer and the tissue were counted using liquid scintillation counter and the results were expressed as a percentage of the total radioactivity.

When slices were exposed to hypoxia for 20min, (3)**H -5-HT release was markedly decreased and are bound release of (3)**H -5-HT was observed on the post-hypoxic period. NMDA(1 mM) increased (3)**H -5-HT release in the control group. NMDA also

increased rebound release of (3)**H -5-HT in post-hypoxic period and prevented hypoxia-induced decrease of (3)**H -5-HT release. When 2-amino-5-phosphonovaleric acid(APV, 307M or 607M ) were added to the incubation media, NMDA-induced increase of (3)**H -5-HT release was blocked dose-dependently. The rebound release of (3)**H -5-HT during post-hypoxic period was also blocked by APV.

These results suggest that the spontaneous release of (3)**H -5-HT decreases during hypoxic period, but 20min hypoxic exposure causes rebound increase of (3)**H -5-HT release during post-hypoxic period which is mediated by the increased activity of the NMDA receptor.