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Objective To investigate the acute effect of apolipoprotein E(apoE)on the intracellular free Ca~(2+) of rat cortical neurons. Methods The intracellular resting calcium level in cultured primary rat cortical neurons was measured by using confocal fluorescent imaging technique. MK-801, an N-methyl-D-aspartate (NMDA) receptor noncom-petitive antagonist, was employed to test potential function of apoE4 through blocking NMDN receptor. Results Acute application of apoE4, but not apoE3, significantly increased the resting [Ca~(2+)] i in a dose-and time-dependent manner (P <0. 01 or P <0. 05) , and MK-801 partly blocked the apoE4-induced elevation of resting [Ca~(2+)]i (P <0. 05 or P <0. 01). Conclusion Acute administration of ApoE4 disturbs calcium homeostasis, and the activation of NMDA receptor may play a critical role in the intracellular calcium overload induced by apoE4 and neurotoxicity.
ABSTRACT
Objective To investigate the acute effect of apolipoprotein E(apoE)on the intracellular free Ca2+ of rat cortical neurons.Methods The intracellular resting calcium level in cultured primary rat cortical neurons was measured by using confocal fluorescent imaging technique. MK-801,an N-methyl-D-aspartate (NMDA) receptor noncompetitive antagonist,was employed to test potential function of apoE4 through blocking NMDN receptor. ResultsAcute application of apoE4,but not apoE3,significantly increased the resting [Ca2+]i in a dose-and time-dependent manner (P
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AIM:To study the effects and mechanisms of a novel sulfonylureous compound 1 ｛4 [2 (4 bromobenzenesulfonaminoethyl)phenylsufonyl｝ 3 (trans 4 methylcyclohexyl) urea, G004, on antithrombosis. METHODS: The influence of compound G004 on the vasoconstrictor action, platelet aggregation and thrombosis formation was studied. The effects of compound G004 on the tail vein bleeding time in mice was examined. The influence of compound G004 on the release of prostanglandin I 2 and thromboxan A 2 from ECV304 cells was investigated. The measurement of cytosolic free Ca 2+ in attached ECV304 cells loaded with Fluo3/AM was carried out. RESULTS: Compound G004 did not inhibit the contraction of rat aorta rings induced by norepinephrine or potassium chloride, but potently inhibit human platelet aggregation challenged by arachidonic acid and adenosine diphosphate. Compound G004 significantly prolonged the tail vein bleeding time in mice and occlusion time of carotid artery in experimentally thrombotic rats. Compound G004 reduced mice mortality induced by the collagen plus epinephrine in a dose dependent manner. Compound G004 enhanced PGI 2 release and reduced TXA 2 secretion from ECV304 cells. G004 had no effect on the increase of cytosolic free Ca 2+ induced by patassium chloride. CONCLUSION: The compound G004 has a remarkable antithrombotic effect in vivo. Its active mechanism may be attributed to inhibition of platelet aggregation, enhancing PGI 2 generation and decreasing TXA 2 secretion from human umbilical vein endothelium.
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AIM To observe the effect of quateranary ammonium salt derivative (F 2) of haloperidol on calcium level in vascular smooth muscle cells (VSMCs) isolated from thoracic aortas of rat. METHODS VSMCs were loaded with Fluo 3 AM, a calcium sensitive fluorescent dye, and [Ca 2+ ] i was determined by the use of laser scanning confocal microscope (LCSM). RESULTS In Ca 2+ (1 26 mmol?L -1 ) bath solution, intracellular calcium fluorescent intensity (FI) in VSMCs was increased when application with 30 mmol?L -1 KCl, and then was inhibited after addition with F 2. The FI of the ASMCs with different concentrations of F 2 (0 1,1,10,100 ?mmol?L -1 )within three minutes were 64%?9%( n =16, P
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AIM To get an insight into intracellular signaling steps, a very early step in the signaling cascade, the biphasic Ca 2+ elicited by 5 HT in rat stomach fundus smooth muscle cells was investigated. METHODS Cells were cultured and loaded with Fluo 3 AM. [Ca 2+ ] i was measured by fluorescent intensity (FI) in each cell with confocal microscopy. RESULTS The resting FI level of SFSMC was 264?15. Stimulation of SFSMCs by 5 HT produced an elevation of [Ca 2+ ] i; Depletion of external Ca 2+ by addition of EGTA led to a significant attenuation of [Ca 2+ ] i change induced by 5 HT; Pre treatment of SFSMCs with ryanodine (10 ?mol?L -1 , 5 min) in D Hanks, the effect of 5 HT was completely inhibited; The stimulation of SFSMCs by 5 HT was partly attenuated by miaserin(10 ?mol?L -1 ), however, L type Ca 2+ channel antagonist lacidipine and G protein inhibitor NEM completely abolished the increase of [Ca 2+ ] i mediated by 5 HT; 5 HT mediated Ca 2+ release was reduced by phospholipase C specific inhibitor compound 48/80(1 2 ?g?ml -1 ); When protein kinase C was activated by phorbol 12 myristate 13 acetate (PMA 0 1 ?mol?L -1 , 5 min) the effect of 5 HT was inhibited, and the inhibitory effect of PMA was reversed by D sphingosine, a PKC inhibitor. CONCLUSION Our data suggest that G protein coupled 5 HT 2B receptor in the rat stomach fundus modulates 5 HT stimulated Ca 2+ increase, and it is coupled to calcium influx through L type calcium channels, and also intracellular calcium release by the opening of ryanodine receptor. The 5 HT 2B receptor mediated signal of 5 HT is transduced by PLC and PKC.