When the intact rabbit iris-ciliary body was incubated in Tyrode's solution containing 10(-4) M hydrogen peroxide, the concentration of hydrogen peroxide remaining in solution diminished rapidly. The iris-ciliary body was homogenized and centrifuged at 100 000 g. It was observed that the hydrogen peroxide detoxification activity was resident primarily in the 100 000 g supernatant and not in the pellet. The hydrogen peroxide detoxification activity was found to be inactivated by heat and to be non-dialyzable. Gel filtration chromatography experiments revealed that breakdown of hydrogen peroxide by the ciliary body was not a 'bulk effect' due to generalized oxidation of tissue constituents. In fact, the active principle, localized by gel filtration chromatography, identified closely with catalase. These observations indicate that catalase within the iris-ciliary body enables the tissue to detoxify hydrogen peroxide from solution. Catalase might protect the iris-ciliary body from damage by hydrogen peroxide generated by normal physiological or pathophysiological conditions.
To better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity.
The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ∼40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ;10 µM) and protein kinase G (KT5823;1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
