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M. Coca–Prados, J. Geibel, S. Ghosh; Somatostatin Enhances Phosphorylation of Protein Kinase Akt at Residue Ser473 and of eNOS at Residue Ser617 in the Bovine Ciliary Epithelium: Possible Implication of the PI3K–Akt Signaling Pathway During SST–Mediated Inhibition of the Na+/H+–Exchanger . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2947.
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The Na+/H+–exchanger is implicated in aqueous humor secretion by the ciliary epithelium (CE). Somatostatin (SST) and natriuretic peptides (NPs) inhibit the Na+/H+–exchange activity of rat and bovine CE. The purpose of this work is to study for upstream events in SST–mediated inhibition of the Na+/H+–exchanger.
Dissected ciliary processes from bovine eyes were treated for 10–15 min at 37oC in the presence or absence of increasing concentrations of SST (10–8 to 10–4 M). Wild type eNOS, phospho–eNOS and phospho–Akt were analyzed by Western blot with specific antibodies to eNOS, phospho–eNOS (Ser617, Ser635 and S1179) and Akt (Ser473). Antibodies to prohormone convertases (PC1 and PC2) and eNOS were also used by indirect immunofluorescence on cryostat sections of bovine ciliary processes. RT–PCR or Northerns were used to determine expression of SST and SST receptors (SSTRs) and eNOS. Radioimmunoassay (RIA) was used to measure SST.
SST and SSTRs are coexpressed in the CE. SST or SST–like material is detected by radioimmunoassay in tissue extracts from CE, and in aqueous humor fluid. PC1 and PC2 (which process pro–STT into SST–14 and SST–28), and eNOS labeled the NPE cell layer. These results indicate that eNOS is expressed in the NPE cell layer of the CE (in addition to the vascular endothelial cells), and there is a differential maturation of pro–SST peptides within the CE. Increasing concentrations of SST elicited increased phosphorylation in residue Ser473 of the protein kinase Akt. This response was efficiently blocked in the presence LY294002 (50µM), an inhibitor of the phosphoinositide–3 kinase (PI3K). Furthermore, SST also enhanced phosphorylation at residue Ser617 of eNOS. In contrast two different sites of eNOS at Ser635 and Ser1179 were not phosphorylated.
Collectively, these results suggest that among the endocrine actions of SST in the CE figure the inhibition of the Na+/H+–exchanger. This action is exerted in part by upstream and downstream cell signaling events in the PI3K/Akt pathway. An upstream event is the phosphorylation of protein kinase Akt at Ser473 and the phosphorylation of eNOS at Ser617 which is a modulator of Ca2+/calmodulin sensitivity of eNOS in nitric oxide (NO) release. NO stimulates soluble GC to form cGMP by the NO/cGMP pathway. cGMP may be therefore a common downstream signal of SST and NPs–induced inhibition of the Na+/H+–exchanger in the CE.
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