The predominant risk factor for the progression of glaucoma is an increase in IOP, mediated via a reduction in aqueous outflow through the conventional (trabecular meshwork and Schlemm's canal) outflow pathway. Current IOP lowering pharmacological strategies target the uveoscleral (nonconventional) outflow pathway or aqueous humor production; however, to date no therapy that primarily targets the conventional pathway exists. Nitric oxide (NO) is an intracellular signaling molecule produced by endogenous NO synthases, well-known for its key role in vasodilation, through its action on smooth muscle cells. Under physiological conditions, NO mediates a multitude of diverse ocular effects, including maintenance of IOP. Nitric oxide donors have been shown to mediate IOP-lowering effects in both preclinical models and clinical studies, primarily through cell volume and contractility changes in the conventional outflow tissues. This review is focused on evaluating the current knowledge of the role and mechanism of action of endogenous NO and NO donors in IOP regulation. Data on key additional functions of NO in glaucoma pathology (i.e., ocular blood flow and effects on optic neuropathy) are also summarized. The potential for future therapeutic application of NO in the treatment of glaucoma is then discussed.
Electroneutral NaCl absorption mediated by Na؉ /H ؉ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na ؉ homeostasis.cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. In vitro conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 in vitro, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.The rapid elevation of intestinal cAMP and cGMP levels by activation of adenylate cyclase and guanylate cyclase, respectively, inhibits intestinal NaCl absorption, either moderately as part of normal digestive physiology or excessively in diarrheal diseases. Some details of the mechanisms of acute regulation of intestinal NaCl absorption by cAMP are understood. Hormones such as vasoactive intestinal peptide or secretin and enterotoxins such as cholera toxin activate adenylate cyclase and increase cellular cAMP content. According to the current model, based on studies in PS120 fibroblasts and the polarized OK 1 renal proximal tubule cell line, acute elevation of cAMP inhibits NHE3 by stimulating its endocytosis plus decreasing its exocytosis and, additionally, by decreasing the NHE3 turnover number (1-4). NHE3 and cAMP-dependent protein kinase type II (PKAII) are part of the same signaling complex that is scaffolded by either of two brush border (BB)-associated PDZ domain containing proteins, NHERF1 (also called NHERF or EBP50) or NHERF2 (also called E3KARP) (5, 6). NHERF1/ NHERF2 each contain two homologous PDZ domains (PDZ1 and PDZ2) and an ERM (ezrin-radixin-moesin) binding domain, which anchors NHERF and its binding partners to the actin cytoskeleton via NHERF binding to ezrin. Ezrin binds both NHERF1/NHERF2 and PKAII and acts as a low affinity cAMP kinase-anchoring protein (AKAP), positioning PKAII so it can phosphorylate NHE3, which is required for cAMP inhibition of NHE3 (1, 6,7).In some cases, cGMP regulates intracellular events by mechanisms analogous to those demonstrated for cAMP. However, the effects of cGMP in the small intestine are not fully elucidated. The intrinsic ileal peptide guanylin and the Escherichia coli heat-stable enterotoxin (STa) both bind to the same BB receptor, guanylate cyclase-C, and within minutes increase intracellular cGMP content (8). STa, guanylin, and cGMP all rapidly i...
Cell biological approaches were used to examine the location and function of the brush border (BB) Na(+)/H(+) exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.
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