Electron probe X-ray microanalysis of intact pathway for human aqueous humor outflow

Abstract

Intraocular pressure (IOP) is regulated by the resistance to outflow of the eye's aqueous humor. Elevated resistance raises IOP and can cause glaucoma. Despite the importance of outflow resistance, its site and regulation are unclear. The small size, complex geometry, and relative inaccessibility of the outflow pathway have limited study to whole animal, whole eye, or anterior-segment preparations, or isolated cells. We now report measuring elemental contents of the heterogeneous cell types within the intact human trabecular outflow pathway using electron-probe X-ray microanalysis. Baseline contents of Na(+), K(+), Cl(-), and P and volume (monitored as Na+K contents) were comparable to those of epithelial cells previously studied. Elemental contents and volume were altered by ouabain to block Na(+)-K(+)-activated ATPase and by hypotonicity to trigger a regulatory volume decrease (RVD). Previous results with isolated trabecular meshwork (TM) cells had disagreed whether TM cells express an RVD. In the intact tissue, we found that all cells, including TM cells, displayed a regulatory solute release consistent with an RVD. Selective agonists of A(1) and A(2) adenosine receptors (ARs), which exert opposite effects on IOP, produced similar effects on juxtacanalicular (JCT) cells, previously inaccessible to functional study, but not on Schlemm's canal cells that adjoin the JCT. The results obtained with hypotonicity and AR agonists indicate the potential of this approach to dissect physiological mechanisms in an area that is extremely difficult to study functionally and demonstrate the utility of electron microprobe analysis in studying the cellular physiology of the human trabecular outflow pathway in situ.

The Glaucoma-associated Olfactomedin Domain of Myocilin Is a Novel Calcium Binding Protein

“…The fluorescence of the nanomolar affinity Ca 2ϩ chelator Quin-2 (29) was then measured under native and denaturing conditions. 4 Reactions containing 8 M myoc-OLF or myoc-OLF(D380A), 150 M Quin-2, and either 0 or 1.4 M GndHCl in 10 mM Hepes, pH 7.5 buffer were added to Bio-One Fluotrac 200 (Grenier) 96 half-well plates. Fluorescence emission was measured on a Biotek Synergy 2 plate reader (excitation, 360/40 nm; emission, 528/20 nm).…”

Ocular Purine Receptors as Drug Targets in the Eye

“…For example, activation of A 1 AR or A 2A AR in juxtacanalicular tissue cells increased the concentrations of Cl -and K + and the cell volume measured by electron probe X-ray microanalysis. 47 The intracellular ionic composition of cells in the inner wall of Schlemm's canal was similarly affected by activating the A 2A AR, but not the A 1 AR. The differential effects of AR agonists to alter the resistance to outflow of aqueous humor have also been studied.…”

Endogenous Regulation of Human Schlemm's Canal Cell Volume by Nitric Oxide Signaling

“…Recent studies using electron probe x-ray microanalysis in outflow pathway tissue determined that cells from the inner wall of the SC experienced a regulatory volume decrease in response to hypotonic challenge. 30 Because resistance to aqueous humor outflow also resides in the inner wall of the SC, we hypothesized that endogenous NO regulates SC cell volume. Additionally, we tested the involvement of soluble guanylyl cyclase (sGC), cGMP, protein kinase G (PKG), and the BK Ca channel in the NO-induced decreases in SC cell volume.…”