Regulation of Nitric Oxide (NO) On the Chloride (Cl–) secretion across the porcine Ciliary Body Epithelium (CBE) via both cgmp–dependent and –independent pathway

Purpose: Emerging evidences suggested that NO signalling might modulate aqueous humour formation (AHF). In this work, the effects of NO related reagents on the trans–CBE Cl– transport were studied in a modified Ussing chamber. Methods: Freshly dissected porcine CBE were mounted on chambers. Paired preparations from the same eye with comparable electrical parameters were enrolled for the study of unidirectional Cl– transport under short–circuited condition. Stromal–to–Aqueous flux (Jsa) was obtained in one of the paired tissue while Aqueous–to–Stromal flux (Jas) was obtained in the conjugate tissue. The difference between Jsa and Jas was the net Cl– transport. Results: The basal net Cl– transport was 0.96±0.13 µEq/hr.cm2 (Jsa > Jas, P<0.01, n=36). 8–pCPT–cGMP, a cGMP analog (100µM, aqueous addition) increased the Cl– secretion by 31% (p<0.01, n=10). However, NO donor, SNP (100µM, bilateral addition) reduced the Cl– secretion by 44% (p<0.01, n=6) while another NO donor, SNAP (100µM, aqueous addition) produced no significant change (p>0.05, n=7). With the pre–treatment of ODQ, a sGC inhibitor (10µM, bilateral addition), the subsequent reduction of the Cl– secretion induced by SNP and SNAP were increased to 63% (p<0.05, n=4) and 25% (p<0.05, n=4), respectively. These results indicated that NO might exert two opposite effects on its modulation on the Cl– secretion across the porcine CBE. In that, the Cl– secretion was enhanced via a cGMP–dependent pathway but inhibited via a cGMP–independent pathway. As the Isc changes induced by the above NO related reagents correlated with the changes in the Cl– secretion, they were used as indicators for our screening test for the possible candidates of the cGMP–independent pathway. With the NO donors alone, SNP inhibited the Isc by 46% (p<0.01, n=22) while SNAP insignificantly inhibited the Isc by 4% (p>0.05, n=20). After the pre–treatment of ABT, an inhibitor of cytochrome P450 enzyme (1mM, bilateral addition), the inhibition of NO donors on the Isc was reversed. In that, SNP did not significantly reduced the Isc (–11%, p>0.05, n=8) while SNAP increased the Isc by 20% (p<0.05, n=7). These results indicated that a cytochrome P450 pathway may be responsible for the inhibition of NO on the Cl– secretion. Conclusions: NO may modulate the AHF via both cGMP–dependent and –independent mechanism. The cGMP–independent mechanism of NO that inhibited the Cl– secretion across the porcine CBE may be mediated via a cytochrome P450 pathway.