ISSN 100727626 CN 1123870ΠQ 2007 5 Chinese Journal of Biochemistry and Molecular Biology 23 (5) :410 414 PKC CaMK Ng 3,,,,,, (, 110001) (long2term potentiation, LTP) C (protein kinase c, PKC) Ca 2 + 2 ( Ca 2 + 2 calmodulin dependent protein kinase, CaMK ) (neurogranin, Ng), Wistar, AlCl 3 3, ; (population spike,ps) LTP ; Takai PKC ; Western CaMK Ng,, PKC, ( P < 0101) ;, CaM, ( P < 0105) ;, Ng, ( P < 0105) : PKC Ng CaMK Ng CaM Ng, Ca 2 + 2CaM CaMK, LTP, ; C(PKC) ; Ca 2 + 2 (CaMK ) ; (Ng) ; R995 Effect of Chronic Aluminum Exposure on PKC, CaMK and Neurogranin in Hippocampus of Rat XING Wei, WANG Biao, HAO Feng2Jin, XU Jin2Hua, ZHAO Yan, LIU Su2Yuan, SHI Li2De 3 ( Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, China Medical University, Shenyang 110001, China) Abstract To investigate the mechanism of aluminum2induced impairments to learning and memory, the effects of chronic aluminum exposure on long term potentiation (LTP) were studied The activity of protein kinase C ( PKC) was determined and the Effect of aluminum on protein expressions of Ca 2 + Πcalmodulin dependent protein kinase (CaMK ) and neurogranin (Ng) were detected The ablactated Wistar rats were used to establish the chronic aluminium exposure models by the gavage of AlCl 3 of different concentrations in the drinking water And 3 months later, the concentrations of aluminum in brain and blood were measured, PS (population spilke) LTP in hippocampus of the rat was recorded, and then the activity of PKC was determined by modified Takai Method Western blotting was used to determine the protein expressions of CaMK and Ng in hippocampus The PKC activity of AlCl 3 exposure groups is obviously lower than that of control groups ( P < 0101) The protein expression of Ng in the hippocampi of AlCl 3 2exposure groups falls down comparing with that of control groups in a dose dependent manner ( P < 0105) The protein expression of CaMK in the : 2006211201, : 2007201231 (No 30371229) 3 Tel : 13002427163,E2mail : nietzschewb @yahoo com cn Received : November 1,2006 ;Accepted : January 31,2007 Supported by National Natural Science Foundation of China (No 30371229) 3 Corresponding author Tel : 13002427163,E2mail : nietzschewb @yahoo com cn
5 : PKC CaMK Ng 411 hippocampi of AlCl 3 2exposure groups is lower than that of control groups in a dose dependent manner too ( P < 0105) Chronic aluminum exposure can reduce not only the activity of PKC but also the protein expressions of CaMK and Ng and the phosphorylation level of Ng, thus affecting the affinity between Ng and calmodulin (CaM) The adjustment and modulation of Ca 2 + 2CaM complex to CaMK were also impacted, inhibiting the formation of LTP and damaging learning and memory Key words aluminum ; protein kinase C ( PKC) ; calmodulin2dependent protein kinase ( CaMK ) ; neurogranin (Ng) ; learning and memory 113, [3 ],, CA3 Schaffer ( : 313 mm ; 318 mm ; 318 mm), [1 ] : ( high2frequency (neurogranin,ng) stimulation, HFS) 1 2 m 5 20,Ng M, 3 molπl KCl ; Ng, CA1 ( : 313 mm ; 115 mm ;, Ng ), C (PKC) Schaffer CA1 (CaM), (population spike, PS) PS 30 min min 1 ( 25 V, 012,, ms ) PS, (long2term potentiation,ltp) (long2term depression,ltd) LTP Schaffer [2 ] ( CaMK ), (100 HZ 5 s), min CaMK, ( ) PS Ng CaMK PKC LTP, PS (,? 10 %, P < 0105) 30 min,, LTP, ( long2term PKC CaMK Ng depression, LTD), 1 111 [4 ] Wistar ( 11411 ) ( LKB ) (Beckman, ) CaMK, 1 9 ( 10 ( Sigma ) mmolπl Tris2HCI, ph715, 1 mmolπl EDTA, 1 mmolπl (HRP ) ( ) EGTA, 1 mmolπl PMSF, 100 mmolπl NaCl, 50 mmolπl Chemilmager5500 V2103 ( ) [ 2 32 P]2ATP( NaF,1 mmolπl Na 3 VO 4, 1 mmolπl PMSF,1 gπml ), 112 30 g Witar, 4, 10, 1 h ;, 1 9 012 % 014 % 016 % 3 (2 mmolπl EDTA,10 mmolπl EGTA,20 mmolπl Tris2 PS (100 %) HFS PS 114 PKC,1 gπml,1 gπml,50 mmolπl 2,1 mmolπl,0109 % Brij235) ( 6 s 6),4, 27 000 g HCl, ph 715 1 % Triton X - 100, 1 mmolπl
412 23 PMSF,1 gπml,1 gπml,1 gπml,50 mmolπl 2 ), 2 h ( 6 s 6),4, 27 000 g 1 h,, Beckman 014 % AlCl 3 71107 11 40 3 10114 1180 3 115 Western CaMK Ng 016 % AlCl 3 86198 13 57 3 # 11107 1162 3 [5 ],, SDS2PAGE ; ( P < 0105), 012 % 014 % ( P < 0101),014 % 016 %, 11412 PKC 20 Table 1 l, [ 2 32 P] 30 l (25 mmolπl 10 Table 1 Aluminum concentration comparisons in blood and l,125 mmolπl ATP 10 l 1 % protamine 2 l,1 molπl Tris2HCl, ph 715, 1 l, H 2 O 7 l), brain between the aluminum exposure and the control ( gx s, n = 10) 30, 10 min, 25 l Whatman Group Aluminum in blood Aluminum in brain Π g L - 1 Π g g - 1 P81, 75 mmolπl 3 Control, 3 min 5 ml 012 % AlCl 3 51125 14 33 70142 9 38 3 6116 0120 9177 1191 3 The aluminum concentrations in blood and brain were determined by atomic absorption spectrophotometer 3 P < 0101 comparing with that of control group, # P < 0101 comparing with that of 012 % AlCl 3 treated group HRP 3, ;, ChemiImager 212 5 500 V 2103 CA1 PS LTP, 100 Fig 1,, PS 116,PS, P < 0105 ;, PS 150 %,,PS 45 min 2 LTP 211 ; LTD, 1 Fig 1 Percentage of PS amplitude enhancements at different times after HFS was applied PS enhancements in aluminum exposure groups were obviously lower than that in control group 213 PKC 0101) Takai, Table 2 PKC ( P < 214 CaMK Ng
5 : PKC CaMK Ng 413 Table 2 PKC activity comparison between groups ( gx s, n = 10) Group Control PKC activityπpmol min - 1 mg - 1 13138 4115 012 % AlCl 3 9184 3184 3 014 % AlCl 3 8135 2132 3 016 % AlCl 3 7156 2106 3 PKC activity was determined by modified Takai method [4 ] 3 P < 0101 comparing with that of control group, CaMK,Western CaMK Ng, CaMK Ng, Fig 2 Fig 2 Effect of aluminum on protein expressions of CaMK and neurogranin This Western blotting shows the relative amount of CaMK and neurogranin Their grey values, which represent their protein contents, were analyzed with ChemiImager 5 500 V 2 103 Software Values are gx s of three experiments CaM, CaM CaMK 3, CaM,PKC LTP (LTP) (LTD), PKC LTP Ng [6 ], LTP,, Ng [12 15 N2 ( ) ] 2D2 (NMDA) Mg 2 + PKC NMDA,Ca 2 +,, Ng CaMK Ca 2 + Ca 2 + : Ng PKC,PKC PKC Ca 2 + Π (calmodulin, CaM)2, Ng CaM (CaM) Ng, LTP - CaMK ;Ng, LTP [7 ],, CaM,CaMK Ca 2 +, PKC ; CaMK [8,9 ] CaMK PKC, Ca 2 + CaMK, LTP Ca 2 +, LTP CaMK 10,, (PSD) CaMK Ca 2 +, CaMK,, Silva [10 ] CaMK, ; Bach [11 ] CaMK 2Asp2286,,,, CaMK, CaMK, LTP, (Ng) 78, calpacitin,ng PKC CaM,Ng CaM, PKC,Ng
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