Limits...
Ontogeny of ATP hydrolysis and isoform expression of the plasma membrane Ca(2+)-ATPase in mouse brain.

Marcos D, Sepulveda MR, Berrocal M, Mata AM - BMC Neurosci (2009)

Bottom Line: Western blots revealed the presence of the four isoforms in all regions, with similar increase in their expression patterns as those seen for the activity profile.Immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in Purkinje cells.Overall, our findings support a close relationship between the ontogeny of PMCA isoforms and specific requirements of Ca2+ during development of different brain areas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain. danielmc@unex.es

ABSTRACT

Background: Plasma membrane Ca2+-ATPases (PMCAs) are high affinity Ca2+ transporters actively involved in intracellular Ca2+ homeostasis. Considering the critical role of Ca2+ signalling in neuronal development and plasticity, we have analyzed PMCA-mediated Ca2+-ATPase activity and PMCA-isoform content in membranes from mouse cortex, hippocampus and cerebellum during postnatal development.

Results: PMCA activity was detected from birth, with a faster evolution in cortex than in hippocampus and cerebellum. Western blots revealed the presence of the four isoforms in all regions, with similar increase in their expression patterns as those seen for the activity profile. Immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in Purkinje cells.

Conclusion: These results show an upregulation of PMCA activity and PMCA isoforms expression during brain development in mouse, with specific localizations mainly in cerebellum. Overall, our findings support a close relationship between the ontogeny of PMCA isoforms and specific requirements of Ca2+ during development of different brain areas.

Show MeSH
Immunodetection of PMCA isoforms in MV from different brain regions. A. MV (20 μg) from cortex, hippocampus and cerebellum at the indicated developmental stages were submitted to electrophoresis, blotted on PVDF membranes and incubated with specific anti-PMCA1, anti-PMCA2, anti-PMCA3 and anti-PMCA4 antibodies. Representative anti-β tubulin immunoreaction as loading control is also shown. B. Quantification of PMCA isoforms (combining all splice variants within each isoform) respect to β tubulin is represented. C. Total PMCA intensity in brain areas obtained from Fig. 2B by adding the band intensities of all isoforms in each stage of development.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2749858&req=5

Figure 2: Immunodetection of PMCA isoforms in MV from different brain regions. A. MV (20 μg) from cortex, hippocampus and cerebellum at the indicated developmental stages were submitted to electrophoresis, blotted on PVDF membranes and incubated with specific anti-PMCA1, anti-PMCA2, anti-PMCA3 and anti-PMCA4 antibodies. Representative anti-β tubulin immunoreaction as loading control is also shown. B. Quantification of PMCA isoforms (combining all splice variants within each isoform) respect to β tubulin is represented. C. Total PMCA intensity in brain areas obtained from Fig. 2B by adding the band intensities of all isoforms in each stage of development.

Mentions: The expression of PMCA isoforms during development was examined in the MV by Western blot using isoform-specific antibodies (Fig. 2A). The anti-PMCA1 antibody stained two protein bands whose expression progressively increased with development until P15, in the three analyzed regions. The intensity of the lower band was higher in cortex and hippocampus, while in cerebellum both variants were similarly expressed. These bands seem to correspond to PMCA1a (130 kDa) and PMCA1b (134 kDa) variants, according to Filoteo et al. [9]. The PMCA2 antibody stained a spectrum of bands around 126-150 kDa in the three regions, whose intensity progressively increased with developmental stage. This was previously observed in developing chick cerebellum [7]. The anti-PMCA3 antibody recognized three main variants whose expression increased with development. The anti-PMCA4 reaction was very strong in the three regions and increased during development, detecting mainly a protein band that could correspond to variant PMCA4b (133 kDa) and faintly the variant 4a (128 kDa) in hippocampus and cerebellum, according to Filoteo et al. [9]. Immunoreaction with β-tubulin (a representative staining from each region is shown) was used as protein loading control for quantification (Fig. 2B). Overall, the largest increase in the expression of total PMCA appeared at P8 stage in cortex and at P15 in the other regions (Fig. 2C), being in good agreement with the kinetic data.


Ontogeny of ATP hydrolysis and isoform expression of the plasma membrane Ca(2+)-ATPase in mouse brain.

Marcos D, Sepulveda MR, Berrocal M, Mata AM - BMC Neurosci (2009)

Immunodetection of PMCA isoforms in MV from different brain regions. A. MV (20 μg) from cortex, hippocampus and cerebellum at the indicated developmental stages were submitted to electrophoresis, blotted on PVDF membranes and incubated with specific anti-PMCA1, anti-PMCA2, anti-PMCA3 and anti-PMCA4 antibodies. Representative anti-β tubulin immunoreaction as loading control is also shown. B. Quantification of PMCA isoforms (combining all splice variants within each isoform) respect to β tubulin is represented. C. Total PMCA intensity in brain areas obtained from Fig. 2B by adding the band intensities of all isoforms in each stage of development.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749858&req=5

Figure 2: Immunodetection of PMCA isoforms in MV from different brain regions. A. MV (20 μg) from cortex, hippocampus and cerebellum at the indicated developmental stages were submitted to electrophoresis, blotted on PVDF membranes and incubated with specific anti-PMCA1, anti-PMCA2, anti-PMCA3 and anti-PMCA4 antibodies. Representative anti-β tubulin immunoreaction as loading control is also shown. B. Quantification of PMCA isoforms (combining all splice variants within each isoform) respect to β tubulin is represented. C. Total PMCA intensity in brain areas obtained from Fig. 2B by adding the band intensities of all isoforms in each stage of development.
Mentions: The expression of PMCA isoforms during development was examined in the MV by Western blot using isoform-specific antibodies (Fig. 2A). The anti-PMCA1 antibody stained two protein bands whose expression progressively increased with development until P15, in the three analyzed regions. The intensity of the lower band was higher in cortex and hippocampus, while in cerebellum both variants were similarly expressed. These bands seem to correspond to PMCA1a (130 kDa) and PMCA1b (134 kDa) variants, according to Filoteo et al. [9]. The PMCA2 antibody stained a spectrum of bands around 126-150 kDa in the three regions, whose intensity progressively increased with developmental stage. This was previously observed in developing chick cerebellum [7]. The anti-PMCA3 antibody recognized three main variants whose expression increased with development. The anti-PMCA4 reaction was very strong in the three regions and increased during development, detecting mainly a protein band that could correspond to variant PMCA4b (133 kDa) and faintly the variant 4a (128 kDa) in hippocampus and cerebellum, according to Filoteo et al. [9]. Immunoreaction with β-tubulin (a representative staining from each region is shown) was used as protein loading control for quantification (Fig. 2B). Overall, the largest increase in the expression of total PMCA appeared at P8 stage in cortex and at P15 in the other regions (Fig. 2C), being in good agreement with the kinetic data.

Bottom Line: Western blots revealed the presence of the four isoforms in all regions, with similar increase in their expression patterns as those seen for the activity profile.Immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in Purkinje cells.Overall, our findings support a close relationship between the ontogeny of PMCA isoforms and specific requirements of Ca2+ during development of different brain areas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain. danielmc@unex.es

ABSTRACT

Background: Plasma membrane Ca2+-ATPases (PMCAs) are high affinity Ca2+ transporters actively involved in intracellular Ca2+ homeostasis. Considering the critical role of Ca2+ signalling in neuronal development and plasticity, we have analyzed PMCA-mediated Ca2+-ATPase activity and PMCA-isoform content in membranes from mouse cortex, hippocampus and cerebellum during postnatal development.

Results: PMCA activity was detected from birth, with a faster evolution in cortex than in hippocampus and cerebellum. Western blots revealed the presence of the four isoforms in all regions, with similar increase in their expression patterns as those seen for the activity profile. Immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in Purkinje cells.

Conclusion: These results show an upregulation of PMCA activity and PMCA isoforms expression during brain development in mouse, with specific localizations mainly in cerebellum. Overall, our findings support a close relationship between the ontogeny of PMCA isoforms and specific requirements of Ca2+ during development of different brain areas.

Show MeSH