Normal homeostatic adjustment of elevated intraocular pressure (IOP) involves remodeling the extracellular matrix (ECM) of the trabecular meshwork (TM). This entails sensing elevated IOP, releasing numerous activated proteinases to degrade existing ECM and concurrent biosynthesis of replacement ECM components. To increase or decrease IOP, the quantity, physical properties and/or organization of new components should be somewhat different from those replaced in order to modify outflow resistance. ECM degradation and replacement biosynthesis in the outflow pathway must be tightly controlled and focused to retain the complex structural organization of the tissue. Recently identified podosome- or invadopodia-like structures (PILS) may aid in the focal degradation of ECM and organization of replacement components.

Although glaucoma is a relatively common blinding disease, most people do not develop glaucoma. A robust intraocular pressure (IOP) homeostatic mechanism keeps ocular pressures within relatively narrow acceptable bounds throughout most peoples' lives. The trabecular meshwork and/or Schlemm's canal inner wall cells respond to sustained IOP elevation and adjust the aqueous humor outflow resistance to restore IOP to acceptable levels. It appears that the cells sense IOP elevations as mechanical stretch or distortion of the actual outflow resistance and respond by initiating a complex extracellular matrix (ECM) turnover process that takes several days to complete. Although considerable information pertinent to this process is available, many aspects of the IOP homeostatic process remain to be elucidated. Components and mechanisms beyond ECM turnover could also be relevant to IOP homeostasis, but will not be addressed in detail here. Known aspects of the IOP homeostasis process as well as possible ways that it might function and impact glaucoma are discussed. Glaucoma Glaucoma is an optic neuropathy characterized by a distinctive pattern of permanent visual field loss. 1,2Optic disk cupping is also a diagnostic parameter. Elevated intraocular pressure (IOP) is the primary risk factor for glaucomatous optic nerve damage and reducing pressure remains the only treatable component of disease progression.2,3 Although glaucoma is a relatively common blinding disease affecting over 67 million persons worldwide, [3][4][5] it is noteworthy that only 2%-8% of people actually develop this disease within their lifetime and most only at advanced ages. The implication of this observation is that some very efficacious mechanism exists to maintain IOP within acceptable ranges throughout the life of most people. 6Intraocular Pressure IOP is maintained primarily by changes in the aqueous humor outflow resistance, which is thought to reside predominantly within the cribriform or juxtacanalicular ( JCT) region of the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC).6-10 Aqueous humor inflow rates are relatively stable and are not pressure dependent, until very high pressures are achieved. 11,12 Although outflow through the alternative or uveoscleral pathway is clearly important, most of the outflow in humans is through the conventional TM/SC route. 2,7,8,12,13 IOP HomeostasisFor our purposes, in this study, we will define IOP homeostasis as corrective adjustments of the aqueous humor outflow resistance, which occur in direct response to sustained pressure changes and which maintain IOP within acceptable physiological ranges.We hypothesize that the flow resistance within the conventional outflow pathway is continually being adjusted with time frames measured in many hours and that sustained pressure changes serve as a guide for the direction and extent of homeostatic resistance modifications. Since the outflow resistance is thought to be comprised primarily of extracellular matrix (ECM) 6,7,9,10,14,15 and sinc...

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X 7 . In this regard, P2X 7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X 7 agonists ATP and 3′-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X 7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X 7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X 7 -mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ ERK2, p38, and JNK1/JNK2) phosphorylation, and that the antioxidants N-acetyl-cysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X 7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.

. Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-B signaling pathways in murine RAW 264.7 macrophages. Am J Physiol Cell Physiol 286: C923-C930, 2004. First published December 18, 2003; 10.1152/ajpcell.00417.2003.-Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X 7. In this regard, after exposure to bacterial LPS, P2X 7 activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X 7 has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-B, we tested the hypothesis that LPS and nucleotides regulate NF-B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X 7-selective ligand 2Ј-3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate (BzATP) results in the cooperative activation of NF-B DNA-binding activity and a sustained attenuation of levels of the NF-B inhibitory protein IB␣. Interestingly, a persistent reduction in IB␣ levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IB␣ protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IB␣ levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-B/ IB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-B and Ras/MEK/ MAPK-associated pathways. nucleotide receptors; mitogen-activated protein kinases; nuclear factor-B; monocytes/macrophages; cytokines ALTHOUGH ATP IS KNOWN to be an important source of intracellular energy, numerous studies have revealed that high concentrations of ATP (5 mM) in the extracellular milieu following tissue damage or platelet degranulation can play a key role in the regulation of biological responses such as inflammation, platelet aggregation, and smooth muscle contraction (13,20,25,38). Nucleotides have been shown to greatly enhance the effects of bacterial LPS on macrophage and monocyte activation by augmenting the production of mediators such as nitric oxide (NO) and other free radicals, in addition to numerous cytokines, such as interleukin (IL)-1␤ and tumor necrosis factor (TNF)-␣ (26, 27, 39, 51, 55). The overproduction of these mediators, although important for the activation of the immune system and for bactericidal effects, can als...