Chi Wai Do

Nucleoside-Derived Antagonists to A3 Adenosine Receptors Lower Mouse Intraocular Pressure and Act across Species

The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models.

Species variation in biology and physiology of the ciliary epithelium: Similarities and differences

Glaucoma is a leading cause of irreversible blindness worldwide. Lowering intraocular pressure (IOP) is the only strategy documented to delay the appearance and retard the progression of vision loss. One major approach for lowering IOP is to slow the rate of aqueous humor formation by the ciliary epithelium. As discussed in the present review, the transport basis for this secretion is largely understood. However, several substantive issues are yet to be resolved, including the integrated regulation of secretion, the functional topography of the ciliary epithelium, and the degree and significance of species variation in aqueous humor inflow. This review discusses species differences in net secretion, particularly of Cl(-) and HCO(3)(-) secretion. Identifying animal models most accurately mimicking aqueous humor formation in the human will facilitate development of future novel initiatives to lower IOP.

Regulation of gap junction coupling in bovine ciliary epithelium

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 +/- 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (R(f)) in recipient to donor cell was 0.47 +/- 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R(f) by approximately 60% to 0.20 +/- 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 microM) also reduced LY dye transfer by approximately 60%, reducing R(f) from 0.41 +/- 0.05 (n = 15) to 0.17 +/- 0.05 (n = 20) after 10 min. Junctional currents were lowered by approximately 50% (n = 6) after 10-min perfusion with 500 microM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 microM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

Characterisation of Cl⁻ transporter and channels in experimentally induced myopic chick eyes.

Background:  Experimental evidence has shown that myopic and hyperopic optical defocus induces thickening and thinning of the choroids, respectively, moving the retina forward and backward toward the plane of focus; however, the underlying mechanism of this phenomenon remains elusive. It has been hypothesised that the change in choroidal thickness might be elicited by the alteration of ion and fluid transport across the retinal pigment epithelium (RPE). Therefore, the aims of the present study were to determine the content of specific Cl‐ transporter/channel mRNA and proteins in chick RPE in a normal, untreated state and in lens‐induced myopia.

Glutathione attenuates nitric oxide-induced retinal lipid and protein changes

Elevated levels of nitric oxide (NO•), a pro‐oxidant that has been associated with numerous retinal diseases, have been implicated in experimental glaucoma models. This study investigated the oxidative effects of sodium nitroprusside (SNP), a nitric oxide donor, on the retinal lipids and proteins and evaluated the potential protective effects of glutathione (GSH).

Childhood exposure to constricted living space: a possible environmental threat for myopia development

People in Hong Kong generally live in a densely populated area and their homes are smaller compared with most other cities worldwide. Interestingly, East Asian cities with high population densities seem to have higher myopia prevalence, but the association between them has not been established. This study investigated whether the crowded habitat in Hong Kong is associated with refractive error among children.

Cyclic Adenosine Monophosphate Activates Retinal Apolipoprotein A1 Expression and Inhibits Myopic Eye Growth.

PURPOSE Apolipoprotein A1 (ApoA1) has been shown to inhibit myopia development in chicks, but the underlying biological mechanism remains unknown. Because ApoA1 interacts with cyclic adenosine monophosphate (cAMP) in many cellular systems, we examined whether this interaction is important in myopia development. METHODS The nonmetabolizable cAMP analogue 8-Bromo-cAMP (8-Br-cAMP) was administered intravitreally to the right eyes of 8-day old chicks for 4 consecutive days. Control eyes received vehicle. Chicks in group 1 received 8-Br-cAMP (0.1 mM or 1 mM) and were fitted with -10 diopter (D) lenses on both eyes, whereas chicks in group 2 (0.1 mM 8-Br-cAMP) wore plano lenses over both eyes. The levels of retinal cAMP and ApoA1 were examined in another two groups of chicks wearing -10 D (group 3) and +10 D lenses (group 4) over their right eyes for 3 days, respectively (plano over left eyes). RESULTS The 8-Br-cAMP significantly inhibited development of lens-induced myopia (group 1: 0.1 mM versus vehicle: +1.71 ± 1.22 D versus -8.00 ± 2.19 D; 1 mM versus vehicle: +1.38 ± 1.34 D versus -9.96 ± 1.14 D, mean ± SEM, P < 0.01 for both); 1 mM, but not 0.1 mM 8-Br-cAMP increased expression of retinal ApoA1 levels in right eyes (P < 0.01). The 8-Br-cAMP had minimal effect on normal eye growth. Both retinal cAMP and ApoA1 levels were significantly increased only in hyperopic eyes (group 4). CONCLUSIONS The 8-Br-cAMP robustly inhibited development of lens-induced myopia. The increase in retinal ApoA1 observed in cAMP-treated and hyperopic eyes suggested a possible interplay between ApoA1 and cAMP in regulating eye growth.

New Insight of Common Regulatory Pathways in Human Trabecular Meshwork Cells in Response to Dexamethasone and Prednisolone Using an Integrated Quantitative Proteomics: SWATH and MRM-HR Mass Spectrometry

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-β pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.