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

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.

Electron microprobe analysis of rabbit ciliary epithelium indicates enhanced secretion posteriorly and enhanced absorption anteriorly.

The rate of aqueous humor formation sequentially across the pigmented (PE) and nonpigmented (NPE) ciliary epithelial cell layers may not be uniform over the epithelial surface. Because of the tissue's small size and complex geometry, this possibility cannot be readily tested by conventional techniques. Rabbit iris-ciliary bodies were divided, incubated, quick-frozen, cryosectioned, and freeze-dried for electron probe X-ray microanalysis of the elemental contents of the PE and NPE cells. We confirmed that preincubation with ouabain to block Na(+),K(+)-ATPase increases Na(+) and decreases K(+) contents far more anteriorly than posteriorly. The anterior and posterior regions were the iridial portion of the primary ciliary processes and the pars plicata, respectively. Following interruption of gap junctions with heptanol, ouabain produced smaller changes in anterior PE cells, possibly reflecting higher Na(+) or K(+) permeability of anterior NPE cells. Inhibiting Na(+) entry selectively with amiloride, benzamil, or dimethylamiloride reduced anterior effects of ouabain by approximately 50%. Regional dependence of net secretion was also assessed with hypotonic stress, which stimulates ciliary epithelial cell regulatory volume decrease (RVD) and net Cl(-) secretion. In contrast to ouabain's actions, the RVD was far more marked posteriorly than anteriorly. These results suggest that 1) enhanced Na(+) reabsorption anteriorly, likely through Na(+) channels and Na(+)/H(+) exchange, mediates the regional dependence of ouabain's actions; and 2) secretion may proceed primarily posteriorly, with secondary processing and reabsorption anteriorly. Stimulation of anterior reabsorption might provide a novel strategy for reducing net secretion.

The ins and outs of aqueous humour secretion.

The intraocular pressure (IOP) reflects a balance between inflow and outflow of aqueous humour. A major strategy in the medical treatment of glaucoma is to reduce inflow and thereby IOP. Understanding the mechanisms and regulation of inflow is thus of clear clinical relevance. Many mechanisms underlying inflow have been identified. The integration and regulation of these mechanisms is less clear. Aqueous humour is secreted across the ciliary epithelium by transferring solute, chiefly NaCl, from the stroma to the posterior chamber of the eye, with water passively following. The epithelium consists of two layers: the pigmented ciliary epithelial (PE) cells abutting the stroma, and the non-pigmented ciliary epithelial (NPE) cells facing the aqueous humour. Gap junctions link adjacent cells within and between these layers. Secretion proceeds in three steps: (1) uptake of NaCl from stroma to PE cells by electroneutral transporters, (2) passage of NaCl from PE to NPE cells through gap junctions, and (3) release of Na+ and Cl- through Na+,K+-activated ATPase and Cl- channels, respectively. Most of our understanding of inflow mechanisms has been obtained by studying in vitro preparations at subcellular, cellular and tissue levels. A particularly productive approach has been the electron probe X-ray microanalysis (EPMA) of the elemental composition of excised ciliary epithelium. This technique permits analysis of adjacent cells within different regions of the ciliary epithelium. EPMA of rabbit preparations has supported the idea that paired activity of Na+/H+ and Cl-/HCO3- antiports can be the dominant mechanism underlying the first step in secretion, stromal NaCl uptake by PE cells. EPMA also indicates that Cl- turnover is faster in the anterior than the posterior region of the epithelium. At the opposite epithelial surface, release of Na+ through Na+,K+-activated ATPase of NPE cells is also greater anteriorly than posteriorly. The accompanying release of Cl- through ion channels is enhanced by agonists of A3 adenosine receptors (ARs). The concepts that paired antiport activity is important in stromal NaCl uptake and that A3ARs modulate NaCl release into the aqueous humour were based on in vitro studies. The potential relevance of these conclusions to in vivo conditions has been tested by measurements of IOP in the living mouse. The results have confirmed the predictions that inhibitors of Na+/H+ antiports lower IOP, and that A3AR agonists and antagonists raise and lower IOP, respectively.

Electron microprobe analysis of ouabain-exposed ciliary epithelium: PE-NPE cell couplets form the functional units.

Aqueous humor is secreted by the bilayered ciliary epithelium. Solutes and water enter the pigmented ciliary epithelial (PE) cell layer, cross gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, and are released into the aqueous humor. Electrical measurements suggest that heptanol reduces transepithelial ion movement by interrupting PE-NPE communication and that gap junctions may be a regulatory site of aqueous humor formation. Several lines of evidence also suggest that net ciliary epithelial transport is strongly region dependent. Divided rabbit iris-ciliary bodies were incubated in chambers under control and experimental conditions, quick-frozen, cryosectioned, and freeze-dried. Elemental intracellular contents of NPE and PE cells were determined by electron probe X-ray microanalysis. With or without heptanol, ouabain produced concentration- and time-dependent changes more markedly in anterior than in posterior epithelium. Without heptanol, there were considerable cell-to-cell variations in Na gain and K loss. However, contiguous NPE and PE cells displayed similar changes, even when nearby cell pairs were little changed by ouabain in aqueous, stromal, or both reservoirs. In contrast, with heptanol present, ouabain added to aqueous or both reservoirs produced much larger changes in NPE than in PE cells. The results indicate that 1) heptanol indeed interrupts PE-NPE junctions, providing an opportunity for electron microprobe analysis of the sidedness of modification of ciliary epithelial secretion; 2) Na and K undergo faster turnover in anterior than in posterior epithelium; and 3) PE-NPE gap junctions differ from PE-PE and NPE-NPE junctions in permitting ionic equilibration between adjoining ouabain-stressed cells.