에탄올이 신경아세포종 B103세포의 Protein Kinase C Isozyme 활성에 미치는 영향

국문 초록 (Abstract)

에탄올이 지속적으로 뇌 신경세포에 미치는 영향을 조사하기 위하여 흰쥐의 신경세포로부터 유래 된 B103 neuroblastoma cell을 사용하여 세포독성이 나타나지 않는 에탄올 농도(0, 50, 100, 200 mM)에서의 1, 2, 8, 18, 24시간 경과에 따라 유도되는 PKC α, γ, ε, ζ isozyme들의 양을 세포질분획과 세포막 분획으로 나누어 Western blot으로 각각 분석하였다. 100 mM의 에탄올 농도에서 분석된 PKC isozyme들 중 PKC-γ는 18시간대의 세포질에서, 그리고 PKC-ε은 8~18시간대의 세포막분획에서 각각 현저한 유도현상을 보였다. PKC-α는 200 mM의 에탄올 첨가 후 18시간과 24시간에 세포질과 세포막 분획에서 모두 대조군의 150%까지 현저한 증가를 나타낸 반면 PKC-ζ는 100, 200 mM 에탄올 농도에서 배양(18, 24시간 동안)한 세포의 세포막분획에서만 유도되었다. 그리고 50, 100, 200 mM의 에탄올 농도에서 24시간동안 배양한 세포질 분획에서 PKC-γ는 농도 의존적으로 감소하여 200 mM의 에탄올 농도에서는 대조군의 47%까지 현저한 감소를 나타내었으며, 세포내에 세포독성을 나타내지 않는 농도 특히 100~200 mM 농도범위의 에탄올을 첨가하여 24시간 동안 지속적으로 배양할 때 PKC-γ 및 ε이 관련된 신호전달체계가 억제됨을 보였다. 이는 에탄올이 PKC isozyme들의 상호간 조절을 통해 신호전달계 또는 신경전달 물질들의 변화에 영향을 줄 수 있음을 시사하며 에탄올의 중추신경계에 미치는 지속적 영향으로 나타나는 행동장애 및 뇌기능의 손상 또는 보호과정에 PKC-isozyme들이 관여할 수 있음을 시사한다.

다국어 초록 (Multilingual Abstract)

It is well known that long-term heavy ethanol intake causes alcoholic dementia, cerebellar degeneracy or Wernicke-Korsakoff syndrome and aggravates the conditions of many other neuro-psychotic disorders. Recently it is indicated that protein kinase C (PKC) plays an important role in the action of ethanol and in the neuro-adaptational mechanisms under chronic ethanol exposure. In order to investigate the effect of ethanol on PKC isoforms levels within the range of not showing any cytotoxicity, B103 neuroblastoma cell line transformed from murine central nervous system was employed and western blot analysis was carried out by using PKC isoform-specific antibodies. The changes of PKC-α, γ, ε and ζ level in the range of ethanol concentration 50~200 mM were examined at the exposure time 1, 2, 8, 18 and 24 hrs in both cytosolic and membrane fraction. A typical ethanol concentration inducing the PKC isozymes was 100 mM, and the transforming time ranges of PKC isozymes could be considered as two different parts to each PKC isoform such as initial (0~2 hrs) and prolonged (8~24 hrs) stages. PKC-γ and PKC-ε were clearly induced during the prolonged stages in cytosol at 18 hrs, and membrane fraction at 8 hrs and 18 hrs, respectively. On the other hand the PKC-α and PKC-ζ isozymes were largely induced in the prolonged stages at 18 hrs and 24 hrs, where the PKC-α isozyme was induced in both cytosol and membrane fractions at 200 mM ethanol concentration while the PKC-ζ isozyme was induced only in the membrane fractions at 100, 200 mM. At 200 mM ethanol concentration of 24 hrs incubation in the prolonged stage, the PKC-α was maximally induced by 150% of the control values whereas the PKC-γ was significantly decreased to 47% of the control values. These results suggest that 100~200 mM ethanol may modulate the signal transduction and neurotransmitter release in the central nervous system through the regulation of PKC isozymes, and the action of these isoforms may act differently each other in the cell.

목차 (Table of Contents)

• Abstract
  
• 서론
  
• 재료 및 방법
  
• 결과 및 고찰
  
• 요약
  
• 문헌

참고문헌 (Reference)

1 "Thiamine utilization in the pathogenesis of alcohol-induced brain damage" 2 273-279, 1994

2 "The role of the protein kinase C in cellular tolerance to ethanol" 271 : 29468-29472, 1996

3 "The protein kinase C family for neuronal signaling" 55-67, 1994

4 "The interaction between risk factors and self-regulation in the development of chronic disease" 81-88, 2001

5 "Signal transduction in environmental neurotoxicity" 21-43, 1998

6 "Rapid colorimetric assay for cellular growth and survival; Application to proliferation and cytotoxicity assays" 55-63, 1983

7 "Protein kinase C isozymes that mediate enhancement of neurite outgrowth by ethanol and phorbol esters in PC12 cells" 85-93, 1993

8 "Protein kinase C and its substrates" 116 : 1-29, 1996

9 "Protein kinase C activation attenuates N-methyl-D-aspartate-induced increases in intracellular calcium in cerebellar granule cells" 1783-1789, 1994

10 "Prenatal and postnatal exposure to ethanol induces changes in the shape of the dendritic spines from hippocampal CA1 pyramidal neurons of the rat" 13-16, 2000

11 "Neurobiology of alcohol abuse" 206-211, 1992

12 "Mutant mice lacking the γ isoform of protein kinase C show decreased behavioral action of ethanol and altered function of γ-aminobutyrate type A receptor" 3658-3662, 1995

13 "Magnetic resonance imaging in alcohol Korsakoff's syndrome; evidence for an association with alcoholic dementia" 479-486, 1996

14 "Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C" 607-258 614, 1992

15 "Induction of the c-jun protooncogene by a protein kinase C-dependent mech-anism during exposure of human epidermal keratinocytes to ethanol" 675-681, 1993

16 "Ethanol-induced effects on expression level and distribution of protein kinase C isoforms in rat liver Golgi apparatus" 114 : 33-43, 1998

17 "Ethanol inhibits kainate responses of glutamate receptors expressed in Xenopus oocytes; role of calcium and protein kinase C" 3162-3171, 1995

18 "Ethanol exposure increases total protein kinase C activity in human lymphocytes" 351-354, 1993

19 "Ethanol and diolein stimulate PKC translocation in astroglial cells" 1037-1042, 1990

20 "Effects of lifelong ethanol consumption on cerebellar layer volumes in AA and ANA rats" 21 : 311-317, 1997

21 "Effect of administered ethanol on protein kinase C in human platelets" 20 : 1503-1506, 1996

22 "Development of glial cells cultured from prenatally alcohol treated rat brain effect of supplementation of the maternal alcohol diet with a grape extract" 313-317, 1996

23 "Chronic ethanol exposure increases levels of protein kinase C -δ and ε protein kinase C-mediated phosphorylation in cultures neural cells" 23428-23432, 1991

24 "Chronic ethanol consumption from euroadaptation to neurodegeneration" 385-431, 1998

25 "Alzheimer's-specific effects of soluble β-amyloid on protein kinase C-α and degradation in human fibroblasts" 5562-5567, 1998

26 "A refined and sensitive method for the quantitation of microgram guantities of protein utilizing the principle of protein-dye binding" 248-254, 1976