ABSTRACT
In order to study the effect of argipressin(4-8)(AVP(4-8)) on the mRNA level and activity of cytidine triphosphate: phosphocholine cytidylyltransferase(CCT) in rat hippocampal neurons, and elucidate it's possible mechanism. Rat hippocampal neurons treated with AVP(4-8) or actinomycin D were incubated with different time periods. The mRNA level of CCT was detected using RT-PCR plus Southern blot, CCT activity was determined by measuring the rate of incorporation of (14)C - phosphocholine into cytidine diphosphate-choline(CDP-choline). It was found that AVP4-8 could upregulate the CCT mRNA in rat hippocampal neurons. ZDC(C)PR, the antagonist of AVP(4-8), could greatly inhibit this upregulation. Using actinomycin D to inhibite the eucaryotic transcription, it was found that the halflife of CCT mRNA could be prolonged by coincubation with AVP(4-8). Meanwhile, AVP(4-8) could also increase CCT activity in rat hippocampal neurons. These results demonstrated that AVP(4-8) upregulated CCT mRNA level and its activity through stabilizing the CCT mRNA in rat hippocampal neurons.
Subject(s)
Arginine Vasopressin/pharmacology , Choline-Phosphate Cytidylyltransferase/metabolism , Hippocampus/drug effects , Neurons/drug effects , Peptide Fragments/pharmacology , Animals , Carbon Radioisotopes , Cells, Cultured , Choline-Phosphate Cytidylyltransferase/genetics , Cytidine Diphosphate Choline/metabolism , Female , Gene Expression Regulation, Enzymologic/drug effects , Hippocampus/cytology , Hippocampus/enzymology , Neurons/enzymology , Phosphorylcholine/metabolism , Pregnancy , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
AIM: To study the localization of CTP: phosphocholine cytidylyltransferase beta isoform (CCTbeta) in rat brain, its expression in insect cells and enzymatic properties. METHODS: Using digoxigenin-labeled CCTbeta probes, in situ hybridization was carried out in rat brain wax sections. CCTbeta was overexpressed in Trichoplusia Ni (Tn) cells using baculovirus expression system. CTP:phosphocholine cytidylyltransferase assay (CT assay) and [3H] metabolic labeling experiment were used to study its activity, properties, and the effect on phosphatidylcholine (PC) synthesis. RESULTS: (1) CCbeta was abundant in CA1, CA2, CA4, and dentate gyrus (DG) region of hippocampus. (2) The content of CCTbeta in transfected Tn cells was over 1 104 times of that in rat brain, and CCTbeta increased the PC synthesis of Tn cells. (3) Hexadecylphosphocholine as well as some ions like Zn2+ and PO3-4 could inhibit the activity of CCTbeta, dCTP was another adaptive substrate of CCTbeta besides CTP. CONCLUSION: CCTbeta showed a similar localization in rat brain with the memory enhancing peptide argipressin (4-8).
Subject(s)
Choline-Phosphate Cytidylyltransferase/metabolism , Hippocampus/enzymology , Moths/metabolism , Animals , Arginine Vasopressin/metabolism , Baculoviridae/genetics , Baculoviridae/metabolism , Brain/enzymology , Choline-Phosphate Cytidylyltransferase/biosynthesis , Choline-Phosphate Cytidylyltransferase/genetics , In Situ Hybridization , Isoenzymes , Moths/genetics , Peptide Fragments/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
ZNC(C)PR can facilitate the learning and memory in rat. Transgenic experiments have revealed that long-term memory depended on cyclic AMP-response element binding protein, CREB. CREB phosphorylation at serine-133 is essential for it's transcriptional activity. Here, it was demonstrated that ZNC(C)PR could induce CREB phosphorylation at serine-133 in both rat hippocampus and rat hippocampus slices. ZDC(C)PR antagnist of ZNC(C)PR , PTX(inhibitor of G(o)/G(I) protein coupled receptor), GF109203x(inhihitor of PKC), PD98059( inhibitor of MAPK ) but not KN-62(inhibitor of CaMKII) could inhibit the phosphorylation of CREB induced by ZNC(C)PR.
ABSTRACT
To understand the mechanism of neurotrophic action of neuropeptide ZNC(C)PR, which could affect growth of C6 cells, fluorescent dye Fluo-3 and confocal laser scanning microscope were used to assay the intracellular calcium in C6 glioma cells. It was found that ZNC(C)PR and it's analogue NLPR could mobilize intracellular calcium in a dose-dependent manner. The ZNC(C)PR antagnist, ZDC(C)PR, could inhibit the process, and the extracellular calcium did not influence it.
