Somatostatin Mediates Na+/H+–Exchanger NHE1 Activity in the Ocular Ciliary Epithelium via the Nitric Oxide (NO)/cGMP Pathway

Abstract

Abstract: : Purpose: Somatostatin (SST) regulates cellular functions including the secretion of hormones. SST has been shown to activate nitric oxide synthase (NOS) in the retina (Vasilaki et al. 2002). SST derives from a polypeptide precursor (pro–SST), and undergoes processing into shorter peptides. In the present study we studied the expression of pro–SST in the ciliary epithelium (CE) and the effects of SST on the intracellular pH (pH_i). Methods: Strips of bovine ciliary processes were incubated with pH sensitive dye BCECF. pH from NPE cells was monitored under HCO₃^– –free conditions using a digital imaging system. After an acid load (20mM NH₄Cl) followed by Na⁺–free solution pH recovery was recorded during subsequent return to Na⁺–containing solution. RT–PCR and Northern were used to determine expression of SST. Radioimmunoassay (RIA) was used to measure SST; and cGMP levels in the presence of the non–specific NOS inhibitors L–NAME and L–NMMA, or the guanylate cyclase inhibitor, ODQ. Results: RT–PCR and Northern blot hybridization, confirmed that the 0.7–kb transcript size of pro–SST is expressed abundantly in the CE. RIA detected SST–28 or a SST–like immunoreactive material in tissue extracts from the bovine CE. SST elicited a dose–dependent attenuation of the rate of pH (pH_i) recovery after acidification, very similar to that reported by natriuretic peptides (NPs) (Fidzinski et al. 2004). Under steady state conditions the rate of pH_i recovery in NPE cells was 0.159±0.007 pH/min (n=86). In the presence of SST 10^–10 M the recovery rate was 0.161±0.01 pH/min (n=38); in the presence of SST 10^–8 M the recovery 0.097±0.008 pH/min (n=34), and finally in the presence of SST 10^–6 M, the recovery rate was 0.054±0.007 pH/min (n=31). SST (from 10^–10 to 10^–6 M) induced up to 6–fold elevation in intracellular cGMP production, within 15 min of treatment, in ciliary explants, and this effect was blocked by L–NAME, L–NMMA or ODQ. Conclusions: SST effect on the Na⁺/H⁺–exchanger can be explained, in part, by its ability to induce the production of cGMP via the NO/cGMP pathway. These observations provide new evidence suggesting that SST and NPs converge through distinct signaling networks on the formation of cGMP to regulate the Na⁺/H⁺–exchanger activity in the CE. We propose that the Na⁺/H⁺–exchanger may serve as a molecular sensor of pH_i and cell volume in aqueous humor secretion and intraocular pressure through the neuroendocrine cell signaling system present in the ciliary processes.

Keywords: ciliary body • nitric oxide • PH regulation/protons