Alba Martin-Gil

Nucleotides in the Eye: Focus on Functional Aspects and Therapeutic Perspectives

The presence and activity of nucleotides and dinucleotides in the physiology of most, if not all, organisms, from bacteria to humans, have been recognized by the scientific community, and the eye is no exception. Nucleotides in the dynamic fluids interact with many ocular structures, such as the tears and aqueous humor. Moreover, high concentrations of nucleotides in these secretions may reflect disease states such as dry eye and glaucoma. Apart from the nucleotide concentration in these fluids, P2 purinergic receptors have been described on the ocular surface (cornea and conjunctiva), anterior pole (ciliary body, trabecular meshwork), and posterior pole (retina). P2X and P2Y purinergic receptors are essential in maintaining the homeostasis of ocular processes, such as tear secretion, aqueous humor production, or retinal modulation. When they are functioning properly, they allow the eye to do its job (to see), but in some cases, a lack or an excess of nucleotides or a malfunction in the corresponding purinergic receptors leads to disease. This Perspective is focused on the nucleotides and dinucleotides and the P2 purinergic receptors in the eye and how they contribute to normal and disease states. We also emphasize the action of nucleotides and their receptors and antagonists as potential therapeutic agents.

Signs and Symptoms of Dry Eye in Keratoconus Patients: A Pilot Study

Abstract Purpose: To compare signs and symptoms of dry eye in keratoconus (KC) patients versus healthy subjects. Methods: A total of 15 KC patients (KC group, n = 15 eyes) and 16 healthy subjects (control group, 16 eyes) were enrolled in this study. The Schirmer I test with no anesthetic, tear break-up time (TBUT), corneal staining characteristics, and ocular surface disease index (OSDI) scores were evaluated for both groups. Impression cytology, combined with/scanning laser confocal microscopy (LCM), was performed to evaluate goblet cell density, mucin cloud height (MCH), and goblet cell layer thickness (CLT). Finally, tear concentrations of di-adenosine tetraphosphate (Ap4A) were assessed. Results were statistically analyzed using Shapiro–Wilk and non-parametric Wilcoxon rank sum tests. Statistical significance was set at p < 0.05. Results: KC patients had lower tear volumes and greater corneal staining than did healthy subjects (p < 0.05). OSDI scores were 44.96 ± 8.65 and 17.78 ± 6.50 for the KC and control groups, respectively (p < 0.05). We found no statistically significant differences in TBUT between groups. Impression cytology revealed lower goblet cell densities in KC group patients versus control group subjects (84.88 ± 32.98 and 128.88 ± 50.60 cells/mm,2 respectively, p < 0.05). There was a statistically significant reduction in MCH and CLT in KC group patients compared with control group subjects. Ap4A tear concentrations were higher in KC group patients than in control group subjects (2.56 ± 1.10 and 0.15 ± 0.12 µM, respectively, p < 0.05). Conclusions: The parameters evaluated in this study indicate that KC patients suffer greater symptoms of dry eye and greater tear instability, primarily due to the decreased mucin production in their tears, than do healthy patients with no KC

Silencing of P2Y2 receptors reduces intraocular pressure in New Zealand rabbits

P2 receptors are involved in the regulation of ocular physiological processes like intraocular pressure (IOP). In the present study, the involvement of P2Y2 receptors in the hypertensive effect of nucleotides was investigated by use of antagonists and of a siRNA designed for the P2Y2 receptor.

2-MeS-β,γ-CCl2-ATP is a Potent Agent for Reducing Intraocular Pressure†

Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydrolyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37 degrees C. In human blood serum, analogues 8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t(1/2) 1 h-30 days). Analogues 8-14 were agonists of the P2Y(1) receptor (EC(50) 0.57-9.54 muM). Ocular administration of most analogues into rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC(50) 95.5 nM). Analogue 9 was more effective at reducing IOP than several common glaucoma drugs and represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.

Involvement of carbonic anhydrases in the ocular hypotensive effect of melatonin analogue 5-MCA-NAT

Abstract:  We have previously demonstrated that melatonin and its analogue, 5‐methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT), reduce intraocular pressure (IOP) in New Zealand rabbits. More recently, we have shown that 5‐MCA‐NAT can also regulate ciliary adrenoceptor gene expression. Like adrenoceptors, carbonic anhydrase (CA) enzymes are involved in aqueous humour secretion by the ocular ciliary epithelium. Moreover, CA enzymes have been reported to be regulated by melatonin. Hence, the aim of this study was to investigate whether the hypotensive effect of 5‐MCA‐NAT is also because of a regulation of CA genes and enzymes. Time course of 5‐MCA‐NAT effect on rabbit IOP was followed for 7 hr every day for up to 144 hr (6 days). 5‐MCA‐NAT reduced IOP, maximally by 51.30 ± 2.41% (at 3 hr), and the hypotensive effect was maintained for up to 96 hr with a single application. IOP studies with 5‐MCA‐NAT plus Trusopt® and immunohistochemical analysis confirmed that CA are molecular targets of 5‐MCA‐NAT. In addition, real‐time quantitative PCR (qPCR) and immunocytochemical assays were performed to determine changes in CA2 (CAII) and CA12 (CAXII) expression in cultured rabbit nonpigmented ciliary epithelial cells (NPE) treated with 5‐MCA‐NAT. NPE cells showed a prominent decrease in both CA, at the mRNA and protein levels. These data confirm that the long‐term hypotensive effect of 5‐MCA‐NAT is also due, to a down‐regulation of CA2 (CAII) and CA12 (CAXII) expression.

Short-term Effect of Scleral Lens on the Dry Eye Biomarkers in Keratoconus.

Purpose To evaluate the most important signs of dry eye, such as osmolarity, inflammation, and diadenosine tetraphosphate (Ap4A) concentration before and after wearing scleral lenses for 8 h in keratoconus patients. Methods A pilot, experimental, short-term study involved 26 keratoconus patients (average age, 36.95 ± 8.95 years). They voluntarily enrolled in the study at the Optometry Clinic of the Faculty of Optics and Optometry in the University Complutense of Madrid. They were divided into two groups: patients with intrastromal corneal ring, the ICRS group, and patients without ICRS, the keratoconus (KC) group. Ocular Surface Disease Index questionnaire, the Schirmer test without anesthesia, tear break-up time, matrix metalloproteinase 9 (MMP-9) concentration, osmolarity, and Ap4A concentration were evaluated before and after wearing a scleral lens for 8 h. Results The patients wore the scleral lenses from 6 to 9 h, with a mean of 7.59 ± 0.73 h. The mean scleral lens sag for all patients was 4310 ± 166.31 &mgr;m, ranging from 4200 &mgr;m to 4800 &mgr;m. No significant changes in the Schirmer test and tear break-up time were found for either group. Ocular Surface Disease Index scores were statistically lower after wearing scleral lenses for both groups (p < 0.05). A significantly lower osmolarity and a significant rise of MMP-9 concentration after wearing scleral lenses were found in both groups (p < 0.05). Diadenosine tetraphosphate concentration was lower after wearing the scleral lens in the KC group (p < 0.05) but no significant difference was found for the ICRS group (p > 0.05). Conclusions Short-term scleral lens wearing improves the symptomatology and some signs of dry eye, such as osmolarity and Ap4A concentration. The increase of MMP-9 concentration could be caused by tear film stagnation and use of preserved saline.

Melatonin and Its Analog 5-Methoxycarbonylamino-N-Acetyltryptamine Potentiate Adrenergic Receptor-Mediated Ocular Hypotensive Effects in Rabbits: Significance for Combination Therapy in Glaucoma

Melatonin is currently considered a promising drug for glaucoma treatment because of its ocular hypotensive and neuroprotective effects. We have investigated the effect of melatonin and its analog 5-methoxycarbonylamino-N-acetyltryptamine, 5-MCA-NAT, on β2/α2A-adrenergic receptor mRNA as well as protein expression in cultured rabbit nonpigmented ciliary epithelial cells. Quantitative polymerase chain reaction and immunocytochemical assays revealed a significant β2-adrenergic receptor downregulation as well as α2A-adrenergic receptor up-regulation of treated cells (P < 0.001, maximal significant effect). In addition, we have studied the effect of these drugs upon the ocular hypotensive action of a nonselective β-adrenergic receptor (timolol) and a selective α2-adrenergic receptor agonist (brimonidine) in normotensive rabbits. Intraocular pressure (IOP) experiments showed that the administration of timolol in rabbits pretreated with melatonin or 5-MCA-NAT evoked an additional IOP reduction of 14.02% ± 5.8% or 16.75% ± 5.48% (P < 0.01) in comparison with rabbits treated with timolol alone for 24 hours. Concerning brimonidine hypotensive action, an additional IOP reduction of 29.26% ± 5.21% or 39.07% ± 5.81% (P < 0.001) was observed in rabbits pretreated with melatonin or 5-MCA-NAT when compared with animals treated with brimonidine alone for 24 hours. Additionally, a sustained potentiating effect of a single dose of 5-MCA-NAT was seen in rabbits treated with brimonidine once daily for up 4 days (extra IOP decrease of 15.57% ± 5.15%, P < 0.05, compared with brimonidine alone). These data confirm the indirect action of melatoninergic compounds on adrenergic receptors and their remarkable effect upon the ocular hypotensive action mainly of α2-adrenergic receptor agonists but also of β-adrenergic antagonists.

Post‐lens tear turbidity and visual quality after scleral lens wear

The aim was to evaluate the turbidity and thickness of the post‐lens tear layer and its effect on visual quality in patients with keratoconus after the beginning of lens wear and before lens removal at the end of eight hours.

The role of dinucleoside polyphosphates on the ocular surface and other eye structures.

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patients tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.

Effect of diinosine polyphosphates on intraocular pressure in normotensive rabbits

The ability of diinosine polyphosphates, diinosine triphosphate (Ip(3)I), diinosine tetraphosphate (Ip(4)I) and diinosine pentaphosphate (Ip(5)I) to modify intraocular pressure in normotensive New Zealand white rabbits was tested. Ip(5)I produced increase in intraocular pressure, while Ip(3)I and Ip(4)I produced a decrease. Ip(4)I was the most effective reducing intraocular pressure inducing a maximal decrease of intraocular pressure to 74.2 ± 2.5% compared with the control value. Dose-response analysis demonstrated a concentration dependent pattern which presented a pD(2) value of 6.19 ± 0.18, equivalent to an EC(50) of 0.63 μM. Regarding the underlying mechanism used by Ip(4)I to reduce intraocular pressure, studies with agonists and antagonists revealed that Ip(4)I reduces intraocular pressure via P2Y receptors in the eye. We suggest that topical application of Ip(4)I to the cornea has therapeutic potential for lowering intraocular pressure, a major risk factor for glaucoma.