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First published online 10 June 2003
doi: 10.1242/jcs.00620

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Differential expression of NO-sensitive guanylyl cyclase subunits during the development of rat cerebellar granule cells: regulation via N-methyl-D-aspartate receptors

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

View larger version (67K): [in a new window]   Fig. 1. Rat cerebellum granule cells cultured for different periods of time. Phase contrast images of 0-day (A), 7-day (B) or 14-day (C) cells in culture. Bar, 50 µm (in all the micrographs).

View larger version (32K): [in a new window]   Fig. 2. RT-PCR showing the expression of NOGCR subunit mRNA in rat cerebellar granule cells (A) and Western blots showing the presence of NOGCR subunits in granule cell extracts (B). + and – indicate PCR reactions performed using equivalent amounts of RNA which had or had not (respectively) undergone previous reverse transcription. Western blotting shows the presence of NOGCR 1 and ß1 subunits in granule cell extracts. 20 µg of soluble cellular extracts were electrophoresed and transferred to PVDF membranes. Blots were incubated with NOGCR, polyclonal antiserum (1:1000) in blocking buffer overnight at 4°C with constant agitation (control), or in the presence of 1 peptide (0.1 µg/ml), ß1 peptide (0.2 µg/ml) or 1 plus ß1 peptide. Once washed (3x10 minutes), the blots were incubated with anti-rabbit-IgG:HRP (1:5000) for 1 hour at 37°C.

View larger version (15K): [in a new window]   Fig. 3. Concentration- (A) and time- (B) dependence curves for DEA/NO-stimulated cGMP accumulation in cerebellar granule cells. 7 DIV cells were washed twice with Locke's solution and incubated in this medium for 1 hour. 0.5 mM IBMX was added to the incubation medium during the last 30 minutes of incubation. The cells were then stimulated with the indicated concentrations of DEA/NO for 10 minutes (A) or with 1 µM DEA/NO for the times indicated (B). Results are expressed as pmol/106 cells corresponding to the mean ± s.e.m. of four experiments performed in triplicate using different cultures. Significant differences from control values (non-DEA/NO stimulated) are indicated: **P<0.01; ***P<0.001.

View larger version (23K): [in a new window]   Fig. 4. (A) 7 DIV granule cells synthesize cGMP in response to NMDA. Cells subjected to different conditions (1 hour incubation with 1 mM L-NAME, 30 minutes incubation with 10 µM ODQ, 30 minutes incubation in a calcium-free medium, 10 minutes incubation with 50 µM AP5, 1 µM MK-801 or 5 µM MK-801) were preincubated with IBMX for 30 minutes and then stimulated for 10 minutes with 100 µM NMDA. Results are expressed as pmol/106 cells and correspond to the mean ± s.e.m. of three experiments performed in triplicate using different cultures. Significant differences from controls are indicated as: ***P<0.001 (NMDA-stimulated).

View larger version (20K): [in a new window]   Fig. 5. Expression pattern of the different NOGCR subunits during the development of cerebellar granule cells in culture (A), and relative contributions of 1 and 2 subunits to total mRNA in granule cells at three developmental stages (B). Total RNA was extracted from freshly isolated cells (FIC), 7 DIV and 14 DIV cells. Equal amounts of mRNA were used for RT reactions and then real time PCR reactions were performed with specific primers as described in Materials and Methods. These results correspond to the mean±s.e.m. of four different experiments performed in triplicate.

View larger version (18K): [in a new window]   Fig. 6. Expression pattern of the different NOGCR subunits during cerebellum development (A) and relative contributions of 1 and 2 subunits to total mRNA in cerebellum at three developmental stages P7, P14 and P21. Equal amounts of mRNA were used for RT reactions and then real time PCR reactions were performed with specific primers as described before. These results correspond to the mean ± s.e.m. of four different experiments performed in triplicate.

View larger version (30K): [in a new window]   Fig. 7. NMDA treatment regulates the expression of 1, 2 and ß1 NOGCR subunits mRNAs. 7 DIV or 14 DIV granule cells were incubated with vehicle (open bars), 100 µM NMDA (striped bars) for 24 hours. When the effect of 50 µM AP5 or 5 µM MK-801 on NMDA action was evaluated they were added to culture media 10 minutes before NMDA addition. After treatment, total RNA was isolated and used for quantitative RT-PCR. Equal amounts of mRNA were used to perform the RT reactions followed by quantitative PCR with the specific primers and probes for 1, 2, ß1 and 18S rRNA. A, B and C represent the relative abundance of 1, ß1 and 2 mRNA (white columns). These results were normalised using the values obtained for 18S rRNA and are the mean±s.e.m. of four experiments performed in triplicate. All the values refer to the amount of mRNA in 7 DIV cells. ***P<0.001; **P<0.01; *P<0.05 are significantly different from the amount of mRNA corresponding to 7 DIV control cells; &&P<0.01 are significantly different from the amount of mRNA corresponding to 14 DIV control cells; +++P<0.001; ++P<0.01; +P<0.05 are significantly different from the amount of mRNA corresponding to NMDA-7 DIV-treated cells; $$P<0.01; $P<0.05 are significantly different from the amount of mRNA corresponding to NMDA-14 DIV-treated cells.

View larger version (28K): [in a new window]   Fig. 8. Effect of NMDA on 1 (A) and ß1 (B) proteins levels in 7 DIV and 14 DIV cells. Cells were incubated for 48 hours in the absence (open bar) or presence of 100 µM NMDA (striped bar). Cells were washed twice with Locke's solution and disrupted as described in Materials and Methods. Equal amount of proteins were loaded into 7.5% acrylamide gel. Proteins were transferred to PVDF membranes and processed as described before. Results are expressed as mean±s.e.m. of four experiments performed with different cell preparations. ***P<0.001; **P<0.01; *P<0.05 are significantly different from the amount of protein corresponding to 7 DIV control cells. ++P<0.01 significantly different from the amount of protein corresponding to 14 DIV control cells.

View larger version (83K): [in a new window]   Fig. 9. Effect of prolonged treatment with NMDA on granule neuron viability. 7 DIV (A-C) or 14 DIV (D-F) cells were incubated with vehicle (control), or 100 µM NMDA for 48 hours and then incubated for 30 minutes in Locke's solution in the presence of the viability/cytotoxicity probes ethidium homodimer (8 µM) and calcein-AM (1 µM). Cell images were taken under the fluorescence microscope by exciting at 495 nm. A 590 nm band pass emission filter was used for the ethidium homodimer (B,E) and one of 530 nm band pass for calcein (A,D). Bar charts (C,E) represent the mean ± s.e.m. of the relative number of EthD-1 labelled cells in different preparations (three different cultures in which four different fields were counted. The total number of cells per field varies from 115 to 200) subjected to the treatment indicated. *P<0.05, **P<0.01 indicates a significant difference from control conditions (non-treated cells).

View larger version (18K): [in a new window]   Fig. 10. Effect of prolonged treatment of 7 DIV granule cells with 100 µM NMDA or 50 µM AP5 on cGMP content in either NO-stimulated or non-stimulated cells (basal response). The indicated concentrations of NMDA (, ) and AP5 (, ) were re-applied every 24 hours with fresh medium. At the required time, cells were washed and preincubated with 0.5 mM IBMX, and then stimulated with either 1 µM DEA/NO (, ) or vehicle (basal, , ) for 10 minutes and their content in cGMP determined. Results are expressed as pmol/106 cells and are the mean ± s.e.m. of four experiments performed in triplicate using different cultures. Values significantly different to those of non-NMDA or AP5-treated cells are indicated: **P< 0.01; ***P< 0.001.

View larger version (15K): [in a new window]   Fig. 11. Dose-response of NMDA on DEA/NO-stimulated cGMP increases in 7 DIV (A) and 14 DIV (B) granule cells. Cells were incubated with the indicated concentrations of NMDA for 48 hours and then stimulated with vehicle (basal response, ) or 1 µM DEA/NO (), and their cGMP content determined as described previously. Results are expressed as mean ± s.e.m. of three experiments performed in triplicate. **P<0.01; ***P<0.001. Corrections for possible cell loss were made by determining the total amount of protein in each well (106 cells=0.1 mg of protein).