A New Method For Measurement Of Release Of SAPP In Rat Hippocampal Brain Slices And Modulation Of Release Of SAPP In The Slices By 5-HT_2C Receptor Mechanism And Other Factors

The work done in our experiment is as follows:Firstly, a new method for measurement of release of soluble( forms of )amyloidβ-protein precursor(sAPP) in rat hippocampal brain slices was developed , which ison the basis of a series of techniques related to hippocampal brain slices and othermethods such as measurement of lactate dehydrogenase (LDH), measurement ofcontent of total protein by bicinchoninic acid assay (BCA), seperating a quantity oftotal hippocampal protein by sodium dodecylsulphate-polyacylamide gelelectrophoresis, detection of the sAPP derivates released into the incubation mediumby western blot analysis. Secondly, the hippocampal slices of rats were incubatedwith various concentration of serotonin, M-110, a 5-HT2C agonist, or L-107, a 5-HT2Cantagonist, sAPP released into the incubation medium were assayed by western blotanalysis assay with monoclonal antibody 22C11 so as to determine whether serotonin,a major neurotransmitter in brain, could modulate the production of the solubleamyloid β-protein precursor (sAPP) by activation of 5-HT2C receptor. Thirdly, L-arginine, L-glutamate and GABA, were used to determine whether these majorexcitatory and inhibitory amino acids in brain, could play roles in modulating theproduction of sAPP. And finally, safflor yellow, was used to determine whether thiscompound would take part in modulating the production of the sAPP.The major results were described briefly as follows:1.The sAPP released into the incubation medium we have detected by westernblot analysis assay with monoclonal antibody 22C 11 all have MW between 97×103Dand 116×103D The new method showed a fine linearity relationship betweenaccumulative grey values of sAPP(y) and total hippocampal protein(x) within the5range from 150-600μg (y=0.5077x-20.721,r=0.9929) This new method is easy tomaster and repeatable.2.Serotonin increased release of sAPP within the concentration range from 10-2μM to 101μM in a concentration-dependent manner and the maximum effectiveconccntration(E.) is 10μM and the increase of sAPP over control is 49.9±5.7%,P<0.01.3.M-110, a 5-HT2C agonist, could increase the release of sAPP within theconcentration range from 1.5×10μM to 1.5×101μM in a concentration-dependentmanner and the maximum effective concentration(E) is 15μM and the increase ofsAPP over control is 98.7±3.9%, P<0.01.4.L-107, a 5-HT2C antagonist, significantly decreased the release of sAPP withinthe concentration range from 1.0×10-9μM to 1.0×102μM in a concentration-dependent manner and the maximum effective concentration(Emax) is 10-6μM and thechange of sAPP over control is -58.4±0.9% P<0.01.5.L-glutamate significantly increased the release of sAPP within theconcentration range from 5.O×10-9μM to 5.O×101μM in a concentration-dependentmanner and the maximum effective concentration(Emax) is 50μM and the increase ofsAPP over control is 49.5±4.90/c, P<0.01.6.L-arginine significantly increased the release of sAPP within the concentrationrange from 1.0×10-9μM to 1.O×10-1μM in a concentration-dependent manner andthe maximum effective concentration(E,.m) is 0.1μM and the increase of sAPP overcontrol is 58.5±4.3%,P<0.01.7.GABA could significantly decrease the release of sAPP within theconcentration range from 1.0×10-9μM to 1.0×101μM in a concentration-dependentmanner and the maximum effective concentration(Emax) is 1.0×10-9 μM and thechange of sAPP over control is -25.9±3.2%, P<0.01.8.Safllor yellow, modulated the release of sAPP in a biphasic manner, that is,within the range of the concentration from 1.0×l0-7μMto 1.0× 10-3μM, the releaseof sAPP was significantly increased in a concentration-dependent manner and the6maximum effective concentration(Em...