Study On The Distribution Of Plasma Membrane Calcium Pump In Rat Cochlea

Objective To study the expression of plasma membrane Ca2+-ATPase isoform 2 (PMCA2) and its alternative splicing at sites A and C in the neonatal rat cochlea.Methods (1) After decapitation, the cochleae from rats postnatal day 3 to postnatal day 4 (P3-P4) were dissected, fixed, embedded, frozen, and sectioned. Meanwhile, the cochlear coils from neonatal rats were isolated and whole mount prepared. Using immunofluorescence staining, the expression of PMCA2 was respectively examined in the cochlear sections and cochlear coils. (2) The total RNAs of basilar membrane (BM, including the organ of Corti, the same below), spiral ganglion (SG), spiral ligament (SL, including stria vascularis, the same below), and the whole cochlea from P3-P4 rats were respectively extracted by Trizol and reverse transcribed to cDNAs, then subjected to primers flanking site A or C in the PMCA2 gene using reverse transcription polymerase chain reaction (RT-PCR). (3) Spiral ganglion tissues isolated from cochleae of P3-P4 rats were cultured in vitro and identified with neuronal marker anti-neurofilament 200 antibody. The expression of PMCA2 was detected by immunofluorescence analyses. The expression of PMCA2 was detected by immunofluorescence analyses. The cultured SGCs were collected and the total RNA was extracted by Trizol and reverse transcribed to cDNA. The site A and C splice variants of PMCA2 were respectively examined by RT-PCR. (4) The BM, SGC, SL and the whole cochlea were respectively isolated and their total proteins were separately extracted and electrophoresed through a 10% SDS-PAGE, then transferred to immobilon polyvinylidene fluoride membrane (PVDF). The membrane was blocked in 5% non-fat milk for 1h at room temperature, incubated with primary rabbit anti-PMCA2 antibody (1:1000) at 4℃overnight, incubated with Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2500) for 1h at room temperature and visualized using the Enhanced Chemiluminescence detection kit.Results (1) PMCA2 was strongly expressed in outer hair cell (OHC) bundles, SG, and stria vascularis (SV), weakly expressed in Reissner's membrane (RM), and occasionally expressed in inner hair cell (IHC) bundles. (2)Variant w/a is the major splice form of PMCA2 present in hair cell bundles, z/b and z/c are the major splice forms of PMCA2 present in SG, and w/a and w/c are the major splice forms of PMCA2 present in SV. In the whole cochlea, variants w, y, and z were detected at site A, and variants a, b, and c were detected at site C. (3) The SGCs grew well with good refraction and showed immunoreactivity for NF-200 antibody. Strong green fluorescence could be seen in cytomembrane, cytoplasm and neuritis of SGCs, showing immunoreactivity for PMCA2 antibody. The results from RT-PCR indicated that PMCA2z was present at splice site A, PMCA2b and PMCA2c were present at splice site C in SGCs. (4) By Western blot, the splice variants at site C of PMCA2 could be identified with PMCA2 antibody. Variant PMCA2a was detected in BM and SV, variant PMCA2b was detected in SG, and variants PMCA2a and PMCA2b were detected in the whole cochlea. These support the results obtained from RT-PCR.Conclusions PMCA2 and its splice variants at sites A and C were differentially expressed in the cochlear tissue of neonatal rat. Objective To study the expression of plasma membrane calcium pump isoforms 1-3 (PMCA1-3) and 5F10 in the developing rat cochlear hair cellsMethods (1) After anaesthetization, the cochleae from rats postnatal day 0 to postnatal day 21 (P0-P21) were dissected, fixed, and the cochlear coils were isolated and used for immunofluorescence staining for PMCA isoforms. After permeated with 0.5% Triton X-100 and blocked in 10% normal goat serum, the samples were respectively incubated with rabbit anti-PMCAl, PMCA2, PMCA3 or 5F10 antibody (that could recognize all four PMCA isoforms)(1:1000) at 4℃overnight, washed with PBS, incubated with Alexa Fluor 488 conjugated goat anti-rabbit antibody(1:400) for 2h at room temperature, washed with PBS, incubated with Alexa Fluor 568 conjugated phalloidin(1:200), washed with PBS, finally viewed using the confocal microscope under the same settings. The fluorescence density of hair cells was measured with Photoshop or Image J and plotted versus ages. (2) The immunogold labeling was also applied to examine the expression of PMCA2 and 5F10 in adult rat cochlear hair cells. Similarly, after fixation, penetration and blocking, the samples were incubated with rabbit anti-PMCA2 or 5F10 antibody (1:50) diluted in 0.05M TBS solution containing 1% BSA and 0.02% Tween 20 (BSA-T20-TBS), washed, incubated with goat anti-rabbit IgG conjugated to 15 nm gold particles diluted 1:20 in BSA-T20-TBS for 2 h at room temperature, washed, stained in aqueous uranyl acetate for 20 min and examined using a transmission electron microscope.Results (1) The PMCA1 expression was absent in IHC and OHC bundles through the whole period. The labeling of PMCA1 in the basolateral membrane of OHCs and IHCs was present within the first 6 days after birth. Thereafter, the labeling in the basolateral membrane of OHCs gradually decreased, till almost disappeared in adulthood. However, the labeling in the basolateral membrane of IHCs gradually increased and reached a steady level in adulthood. (2) The PMCA2 labeling in OHC bundles rapidly increased over the first week after birth, first appeared in the base, then middle, finally apex. The basal labeling was stronger than the apical. At P8, the staining in OHC bundles almost reached adult level and showed no much difference among the apical, middle and basal turns. There was a week expression in IHC bundles within 10 days after birth, but the expression almost disappeared after P13. No staining was observed in the basolateral membrane of OHCs and IHCs at all the time points. (3) The PMCA3 expression was absent in OHC and IHC bundles through the developing period, but present in the basolateral membrane of OHCs and IHCs within 6 days after birth. After P13, PMCA3 labeling almost disappeared in the basolateral membrane of OHCs, but maintained a high level in the basolateral membrane of IHCs into adulthood, although there was a small drop. (4) The labeling of 5F10 in OHC and IHC bundles was similar to the PMCA2 labeling. The 5F10 staining in the basolateral membrane of IHCs and OHCs gradually increased within 6 days after birth, thereafter gradually decreased. The staining in the basolateral membrane of OHCs almost disappeared in adulthood. In contrast, the staining in the basolateral membrane of IHCs still maintained a certain level into adulthood. (5) The results from immunogold labeling for PMCA2 showed that there was no significant difference of the gold particle counts between the three row stereocilium of OHCs. With the PMCA2 labeling, gold particle counts gave a ratio of 3.8/1 for OHC/IHC bundle labeling. With the 5F10 labeling, gold particle counts gave a ratio of 2/1 for OHC/IHC bundle. There was no significant difference for the gold particle counts of PMCA2 labeling between apical and basal OHC bundles.Conclusions PMCA2 appearance in OHC bundle (P0-P6) matches the development of the mechanoelectrical transduction current. Less PMCA2 in IHC bundles than OHC bundles implies smaller Ca2+ load in IHCs. There was no difference of PMCA2 expression between apical and basal OHC bundles in adult rats. IHC basolateral membrane contains both PMCA1 and PMCA3. The variation of 5F10 labeling matches the development of Ca2+ current in IHC soma from P2-P6. Hair cells mainly rely on PMCA to control intracellular Ca2+ level, thus the differential expression of PMCA isoforms may be due to their special functions in hair cells.Objective To study the differential expression of plasma membrane calcium pump isoforms 1-3 (PMCA1-3) in the basilar membrane of P2 and P8 rat cochlea by Western blot. The content of PMCA2 protein in P8 rat cochlear hair cells was also examined.Methods (1) Four rats at P2 and P8 were respectively anesthetized and the basilar membrane was isolated after removing Reissner's membrane and Tectorial membrane. The total proteins of the cochlear coils were extracted and the concentration was examined. The total proteins of 3μg and 5μg were respectively loaded to the gel, electrophoresed through a 7.5% SDS-PAGE and transferred to nitrocellulose membrane. Then the membrane was blocked in 5% non-fat milk for 2 h at room temperature, incubated with primary anti-PMCA2 antibody (1:1000) at 4℃overnight. After washes, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody (1:2000) for 1h at room temperature, washed, and visualized using the Enhanced Chemiluminescence detection kit. (2) After extracted from basilar membrane of P2 and P8 rats, the total proteins of 20μg were respectively loaded to the gel, electrophoresed through a 7.5% SDS-PAGE, transferred to membrane, and incubated with PMCA1-3 antibodies (1:1000). The detailed procedures were described in (1). (3) The total proteins of 5μg,10μg and 20μg from cochlear coils of P8 rats were added to the gel and 100ng,400ng and 800ng BSA proteins were also loaded as reference. After electrophoresis through a 7.5% SDS-PAGE, the gel was separated into two parts. One part (containing 10μg and 20μg total proteins and 100ng, 400ng and 800ng BAS proteins) was used for SYPRO staining and the other part (containing 5μg total proteins) was used for PMCA2 detection by Western blot. The detailed procedures were described in(1).By comparing the location of the bands in the gel stained by SYPRO and in the film by Western blot, the position of the PMCA2 band was located in the gel and its grey value was measured by Photoshop or Image J, then the content of PMCA2 in the band was calculated.Results (1) The expression of PMCA2 was respectively detected in the BM of P2 and P8 rats. The latter was stronger than the former. (2) The expression of PMCA1 was weak in the BM of P2 and P8 rats and there was no obvious difference between the two ages. The content of PMCA2 was rich in the BM of P2 and P8 rats. The expression of PMCA3 was barely detected in the BM of P2 and P8 rats. (3)The content of PMCA2 in the BM of a P8 rat was about 2.5ng.Conclusions There is a different-level expression of PMCA1-3 in the BM of P2 and P8 rat. PMCA1 was weakly expressed. PMCA2 was strongly expressed. PMCA3 was barely expressed. The differential distribution of PMCA1-3 in the BM of neonatal rat indicates that the cochlear hair cells may have special requirements of Ca2+ turning for these PMCA isoforms.