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R. G. Corredor, J. L. Goldberg; Soluble Adenylyl Cyclase (sAC) Promotes Survival and Axon Growth in Retinal Ganglion Cells (RGCs). Invest. Ophthalmol. Vis. Sci. 2010;51(13):2248.
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Mammalian RGCs do not regenerate their axons, and die after optic nerve injury or ischemia, or in glaucoma and other diseases, leading to permanent visual loss. Previous work has shown that an increase in electrical activity or in levels of intracellular cAMP can enhance trophic responsiveness and promote survival and axonal growth of RGCs. However it is not known which adenylyl cyclases are responsible for elevating cAMP in response to electrical stimulation (ES). Here we investigate whether sAC is present in RGCs and promotes survival and axon growth in vitro.
Immunostaining, western blot and RT-PCR were used to investigate expression and localization of sAC in RGCs. Cultured rat postnatal RGCs were tested with the sAC agonist bicarbonate, and the sAC antagonist 2-hydroxyestradiol for effects on survival and axon growth, quantified using time lapse microscopy and axon-tracing at 48 hour in vitro,in the presence or absence of ES and/or the transmembrane adenylyl cyclase(TmAC) activator forskolin. Intracellular levels of cAMP were quantified using ELISA.
ES improved survival of RGCs and promoted axon growth in vitro, even in the presence of forskolin. We detected sAC transcripts in purified RGCs by RT-PCR. By western blotting, one band was detected at approximately 50Kd. Immunostaining showed sAC reactivity in the cytoplasm, nucleus and axons of RGCs. The sAC activator bicarbonate increased intracellular levels of cAMP, stimulated RGC survival, and enhanced axon growth when compared with cells in bicarbonate-free media. Interestingly, bicarbonate also enhanced survival and axon growth of RGCs in the presence of forskolin or the cell-permeable cAMP analog 8-bromo-cAMP. Pharmacological inhibition of sAC dramatically decreased RGC survival and axon growth.
sAC activation increases and sAC inhibition decreases RGC survival and axon growth in vitro. Since electrical stimulation also enhanced RGC survival and axon growth in the presence of TmAC activation, we hypothesize that additional cAMP may be generated in response to activity by the calcium- and bicarbonate-sensitive sAC.
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