Argipressin(4-8) upregulate CTP: phosphocholine cytidylyltransferase in rat hippocampal neurons.

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.

Localization of CCTbeta in rat brain and overexpression in insect cells.

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).

Cloning and Distribution of rRP58, A Novel Neuronal Gene.

EST (AW055733) is a 3'-cDNA fragment specially expressed in rat brain. It is homologous to human and mouse RP58 gene, which is a transcriptional repressor gene. Primers were designed based on these two genes, and then two transcripts of rRP58 gene were got from male SD rat using RT-PCR method. The rRP58 protein is a C(2)H(2) type zinc finger protein, containing POZ domain at N-terminal and zinc finger domain (ZFD) at C-terminal. In addition, there is a highly acidic region between POZ and ZFD. POZ and ZFD were cloned, expressed and purified. Then, the antibodies to POZ and ZFD were prepared, and used to analyze distribution of alternative transcripts of rRP58.

ZNC(C)PR Induces Phosphorylation of CREB in Rat Hippocampus.

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.

Arginine Vasopressin(4-8) Mobilizes Intracellular Calcium in C6 Glioma Cells.

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.

AVP(4-8) Enhances PKC and MAPK Activities in SK-N-SH Cells.

AVP(4-8), one of endogenous metabolite of argipressin(AVP) in brain, can enhance learning and memory. To understand further the molecular mechanism of its function, human neuroblastoma SK-N-SH cell line was chosen as a model to study its signal transduction pathway. Radioligand binding assay showed the existence of binding sites for AVP(4-8) on SK-N-SH cells. The activity of PKC and MAPK in SK cells was significantly enhanced by AVP(4-8), and the enhancement of PKC and MAPK was suppressed by ZDC(C)PR, an antagonist of AVP(4-8).

Cloning of a Gene in Rat Brain Induced to Be Expressed Differently by AVP(4-8) with Differential Display PCR.

As a metabolite of arginine-vasopressin, AVP(4-8) has been shown to have potent memory-enhancing activity and induce a series of physiological and biochemical events in rat brain. The technique known as differential display polymerase chain reaction(DD-PCR)was applied to explore genes induced to be expressed differ-ently by AVP(4-8) in rat hippocampus. Nine different primer pairs were used to perform DD-PCR after reverse transcription, and more than ten differential display fragments were observed. The most remarkable different fragment, dd1, was further cloned and sequenced. Homology searching in Genebank showed that dd1 may be a new gene. Moreover, the results from Northern blot confirmed that dd1 is indeed a gene induced by AVP(4-8).

Effects of ZNC(C)PR and Its Analogs on CNDF mRNA Expression in Rat Brain.

Reverse transcription(RT)-PCR method was used to examine the cholinergic neuron derivative factor (CNDF)mRNA level changes in rat brain after the administration of ZNC(C)PR or its analogs. Glyceraldehyde-3-phophate dehydrogenase (GAPDH)mRNA was co-amplified as an internal control. The CNDF mRNA expression was significantly increased and its peak was reached at 18 h after the administration of ZNC (C)PR (hippocampus, 3.02 fold cortex, 5.33 fold compared to control, P < 0.01). NLPR, agonist of ZNC(C)PR receptor, enhanced the CNDF mRNA transcription to a higher level than ZNC(C)PR. ZDC(C)PR, antagonist of ZNC(C)PR receptor, partially blocked the CNDF transcription when co-administered with ZNC(C)PR. AVP increased the CNDF mRNA expression to lower level than ZNC(C)PR, while oxytocin had no effect on the CNDF transcription. The results suggest that the CNDF gene may be one of the targets of ZNC(C)PR, and its enhancement effect on CNDF mRNA expression was mediated by the ZNC(C)PR receptor.

AVP (4-8) May Stimulate a G Protein-coupled Receptor in Rat Hippocampal Synaptosomal Membranes.

As a metabolite of arginine-vasopressin, AVP(4-8) has been shown to have potent memory-enhancing activity and to induce a series of physiological and biochemical events in rat brain. GTP-binding protein is known to be a revolving stage of transmembrane signal transduction to mediate physiochemical responses of neurotransmitters and neuromodulators. A specific binding site of AVP(4-8) in the rat hippocampal synaptic membranes was identified by radio-receptor assay and after binding to membranes, AVP(4-8) enhanced the binding of Guanosine -5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS), and this enhancement could be completely reversed by the antagonist of AVP(4-8), ZNC(C)PR. Based on the alone results, we suggest that AVP(4-8) exerts its function as neurotransmitter through a G-protein-coupled receptor on the synaptosomal membrane of rat hippocampus.

The Binding Sites of AVP(4-8) in Rat Brain Are Different from the Known Receptors.

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