Localization of cGMP-Dependent Protein Kinase Isoforms in Mouse Eye

To examine the expression of the major isoforms of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (cGK) in mouse eye. Immunohistochemical localization of cGMP in mouse eye cryosections was performed using an anti-cGMP antibody, followed by visualization with indirect fluorescence microscopy. The presence of types Ialpha, Ibeta, and II cGK mRNAs in mouse eye extracts was determined initially by RNase protection analysis. Further localization of cGK I and II mRNAs on cryosections was accomplished by in situ hybridization using digoxigenin-labeled cRNA probes and an alkaline phosphatase-conjugated anti-digoxigenin antibody. Finally, cGK I protein was localized to subcellular areas within the retina using an anti-cGK I-specific primary antibody. In initial immunohistochemical experiments cGMP was present in numerous regions and layers within the eye and retina. Subsequent RNase protection studies demonstrated that cGK Ialpha, Ibeta, and II mRNAs were present in mouse eye and that type Ibeta mRNA were 6.6 and 30 times more abundant than type Ialpha and type II, respectively. By in situ hybridization, cGK I mRNA was localized to photoreceptor inner segments and the ganglion cell and inner nuclear layers of the retina, and lesser amounts were found in the ciliary epithelium, lens, and cornea. The cGK II mRNA expression pattern was similar but not identical with that of cGK I. Finally, within the retina, cGK I protein was most abundant in the inner plexiform layer, with significant amounts in ganglion cells and photoreceptor inner segments as well. The presence of these cGK isoforms in discrete areas throughout the eye suggests multiple roles for the cGMP-dependent signal transduction system in the regulation of physiologic and pathologic ocular processes.