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D Matesic, A Sitaramayya, T Tillen; Expression of Connexin 40 in Mammalian Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):732.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Retinal neurons are known to be extensively coupled, both homologously and heterologously, through gap junction intercellular channels, but little is known about connexin subtypes expressed by mammalian retinal neurons. In addition, retinal gap junction coupling is known to be modulated by both dopamine and NO through connexin phosphorylation, indicating that at least some of the retinal connexins are phosphoproteins. In this work, we explored whether the connexin phosphoprotein subtype, connexin 40, was expressed in mammalian retina. Methods:In immunofluorescent staining experiments, light-adapted bovine and rat retinas were fixed in paraformaldehyde, and 6-8 micron slices permeabilized with methanol were reacted with connexin-specific antibodies for 24 hr. Reaction was visualized with rhodamine or fluorescein secondary antibodies. Puncta were observed using a 100X oil immersion lens. In Western blot experiments, bovine retinal membranes were alkaline-extracted, proteins separated on 12.5% acrylamide gels, transferred to PVDF membranes, and incubated with connexin-specific antibodies. Bands were detected using biotinylated secondary antibodies, AP-conjugated streptavidin and BCIP/NBT. Results:Immunostaining experiments using connexin 40-specific antibodies from two independent sources showed punctate staining between cells in the outer nuclear layer, as well as between cells within a sublayer of the inner nuclear layer. Punctate staining in these two regions was observed in approximately 40-50% of retinal sections scanned. In addition, sparse punctate staining was detected in the GCL/AFL region. No punctate staining was observed in the outer or inner segment layers in either rat or bovine retinas. Double immunostaining of bovine retinas with antibodies to Go, which stains bipolar cells, and to connexin 40, showed little overlap. Western blot analysis of bovine retinas revealed an immunopositive doublet at approximately 40 kD that co-migrated with a doublet present in heart membranes. These immunopositive bands disappeared when antibody was pre-incubated with a peptide containing the connexin sequence to which the antibody was made. Conclusion:These results suggest that connexin 40 is expressed within the neural layers of bovine and rat retinas. Specific cell types expressing connexin 40 remain to be determined.
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