Nitric oxide is an essential mediator for neuronal differentiation of rat primary cortical neuron cells

Abstract

Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.

Keywords: butyrate; neurite outgrowth; nitric oxide; nitric oxide synthase; primary cortical neuron cells.

Fig. 1

The effect of NO scavenger and NOS inhibitor on neurite outgrowth in rat primary cortical neuron cells. (A) Phase-contrast photographs of untreated or 6 mM NMMA (inhibitor of three NOS isoforms) or 1 mM PTIO (NOS scavenger) treated primary cortical neuron cells for 1 day. (B) The number of neurite of primary cortical neuron cells were counted under the microscope for 5 days.

Fig. 2

The effect of NO scavenger and NOS inhibitor on expression levels of p21 and p27 mRNAs in rat primary cortical neuron cells. The primary cortical neuron cells were treated with 6 mM NMMA or 1 mM PTIO as indicated. Cells were harvested at 0.5, 1, 1.5, 2, 3, 5 and 7 days and total RNA was extracted. (A) mRNA expression of p21 was measured by RT-PCR. The PCR product (311 bp) was resolved in 1.5% agarose gel. (B) mRNA expression of p27 was measured by RT-PCR. The PCR product (238 bp) was resolved in 1.5% agarose gel. Con (-): negative control, RNase: RNase treatment, Con (+): positive control, no RT: PCR reaction without reverse transcriptase, Gen: genomic DNA 500 ng.

Fig. 3

The effect of NO scavenger and NOS inhibitor on expression levels of c-jun and c-myc mRNAs in rat primary cortical neuron cells. The primary cortical neuron cells were treated with 6 mM NMMA or 1 mM PTIO as indicated. Cells were harvested at 0.5, 1, 1.5, 2, 3, 5 and 7 days and total RNA was extracted. (A) mRNA expression of c-jun was measured by RT-PCR. The PCR product (775 bp) was resolved in 1.5% agarose gel. (B) mRNA expression of c-myc was measured by RT-PCR and PCR product (342 bp) was resolved in 1.5% agarose gel. Con (-): negative control, RNase: RNase treatment, Con (+): positive control, no RT: PCR reaction without reverse transcriptase, Gen: genomic DNA 500 ng.

Fig. 4

Immunochemical staining analysis of neuronal NOS (nNOS). The primary cortical neuron cells were treated with 6 mM NMMA or 1 mM PTIO as indicated. Cells were fixed by 4% paraformaldehyde and incubated with anit-nNOS monoclonal antibody as describes in materials and methods.

Fig. 5

Measurement of NOS activity in rat primary cortical neuron cells. The primary cortical neuron cells were harvested and prepared cell extracts. NOS activity was measured as described in materials and methods.