W. D. Stamer

Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport

A1 adenosine receptors (ARs) reduce, and A2ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A1AR and A2AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A1, A2Aa and A3AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A1 ARs, CGS 21680 and DPMA for A2AARs, and Cl-IB-MECA and IB-MECA for A3ARs. Stimulation of A1 A2A and A3ARs elevated Ca2+, measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A], A2A and A3AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A1, A2A and A3ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca2+ and volume, but likely by actions on separate cell targets.

Role of Aquaporin-1 in Trabecular Meshwork Cell Homeostasis during Mechanical Strain

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemms canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to beta-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8h (n=4, p<0.05) and 24h (n=6, p<0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n=4, p<0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r(2)=0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.

Relationship between glaucoma and selenium levels in plasma and aqueous humour

Aim: The aim of the study was to compare selenium levels in plasma and aqueous humour in subjects with and without primary open-angle glaucoma (POAG). Methods: Forty-seven POAG cases and 54 controls in this case–control study were recruited from surgery patients at the University Physician’s Ophthalmology Clinic in Tucson, Arizona, USA. Aqueous humour and plasma selenium were determined by high-performance liquid chromatography ion channel plasma mass spectrometry (HPLC ICP-MS). Potential confounders were assessed via a questionnaire. Biological samples were collected and processed at surgery and analysed for selenium content after collection was complete. Outcome measures included the odds of glaucoma in relationship to plasma selenium, aqueous humour selenium, and the ratio of levels of aqueous humour selenium to plasma selenium. Results: Tertile of selenium and its relationship to POAG was examined. After adjustment for common glaucoma risk factors, the odds of glaucoma in the highest tertile of plasma selenium (OR = 11.3; p = 0.03) and the middle tertile of aqueous humour selenium (OR = 0.06; p = 0.02) was significantly associated with glaucoma. Conclusion: Although a causal pathway cannot be inferred from our analysis, our data, added to that of others, suggest that the pathology is selenium-related.

MYOCILIN LEVELS IN PRIMARY OPEN-ANGLE GLAUCOMA AND PSEUDOEXFOLIATION GLAUCOMA HUMAN AQUEOUS HUMOR

PurposeTo determine the concentration of myocilin in primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) aqueous humor. MethodsAqueous humor was collected during surgery from patients with POAG, PEXG, and elective cataract removal (control). Volume-equivalent aqueous samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. Quantification of myocilin levels was performed using Western blots probed with 2 independent N-terminal polyclonal anti-myocilin antibodies (AB1 and AB2) followed by densitometry. Myocilin levels in aqueous humor were quantified by plotting the densitometry readings of the aqueous samples against a recombinant myocilin standard curve. Total protein concentration was determined by Bradford protein assay. Transforming growth factor &bgr; 2 levels were assessed by enzyme-linked immunosorbent assay. ResultsMyocilin levels are significantly elevated in human POAG aqueous humor when compared with control aqueous humor (AB1: 0.66±0.53 ng/&mgr;L vs. 0.23±0.20 ng/&mgr;L, P<0.001; AB2: 0.98±0.59 ng/&mgr;L vs. 0.65±0.5 ng/&mgr;L, P<0.03; mean±SD). Myocilin makes up a larger percent of the total protein in POAG aqueous humor compared with control aqueous (AB1: 0.26±0.20% vs. 0.10±0.20%, P<0.001; AB2: 0.43±0.32% vs. 0.28±0.18%, P<0.05). In contrast to POAG, myocilin levels were not elevated in PEXG aqueous humor when compared with control aqueous humor. No correlation between myocilin and transforming growth factor &bgr; 2 levels was observed. ConclusionsMyocilin is elevated in POAG, but not in PEXG aqueous humor.