María J. Pérez de Lara

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.

Presence of melanopsin in human crystalline lens epithelial cells and its role in melatonin synthesis

ABSTRACT Melanopsin is a non‐image forming photoreceptor known to be present in the retina and it is considered to have light regulated tasks among other functions. In the present work, melanopsin presence in human lens epithelial cells as well as in human lens tissue is described for the first time. Moreover, studying the concentration of melatonin and its synthesising enzyme AANAT proved a clear link between melanopsin activation and the suppression of melatonin synthesis. Melanopsin sensitivity to specific wavelength (465–480 nm, blue) was confirmed after making temporal studies incubating lens epithelial cells under light, red, green, blue and total darkness for 2, 4, 8, 12 h and analysing the concentration of both melatonin and its synthesising enzyme AANAT, discovering that melatonin levels after submitting cells to total darkness are significantly higher to ones submitted to white or specifically blue light (***p < 0.001, n = 6). The involvement of melanopsin in the regulation of melatonin was also determined by using a specific inhibitor AA92593 and by inhibiting melanopsin‐induced phospholipase C activation. Under this situation neither AANAT nor melatonin levels changed under light conditions (n = 4, ***p < 0.001). The discovery of melanopsin in the lens opens the possibility of regulating melatonin synthesis with the corresponding implication as an antioxidant substance. HighlightsHuman lens epithelial cells present melanopsin.Activation of melanopsin with light reduces the synthesis of melatonin.Darkness increases melatonin production and AANAT expression.Melanopsin is activated by blue wavelength and insensitive to red or green light.

Effect of Melatonin and 5-Methoxycarbonylamino-N-Acetyltryptamine on the Intraocular Pressure of Normal and Glaucomatous Mice

Melatonin is a neurohormone that is produced not only by the pineal gland but also by several ocular structures. One of the main physiologic roles of melatonin is the reduction of intraocular pressure (IOP). Using both control C57BL/6J and glaucomatous DBA/2J mice as well as TonoLab tonometry, this study evaluated the effect of melatonin and 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) when glaucomatous pathology was fully established and compared pharmacological behavior in treated mice versus control mice. In addition, 5-MCA-NAT was tested to determine its effects on ameliorating increased IOP in a glaucoma model. The results demonstrate that melatonin and 5-MCA-NAT can reduce IOP in a concentration-dependent manner. The EC50 values for melatonin in control and glaucomatous animals were 34 µM and 50 µM, respectively. Interestingly, melatonin decreased IOP in 19.4% ± 3.7% and 32.6% ± 6.0% of control and glaucomatous mice, respectively, when the animals were studied at age 12 months. 5-MCA-NAT reduced IOP in the same manner and was able to stop IOP progression in glaucomatous mice. Use of melatonin receptor antagonists showed that hypotensive effects were blocked by the MT2 receptor antagonists luzindole and 4-phenyl-2-propionamidotetralin in the case of melatonin and by only 4-phenyl-2-propionamidotetralin in the case of 5-MCA-NAT. In conclusion, melatonin and 5-MCA-NAT can effectively reduce IOP in a glaucoma model, and their hypotensive effects are more profound in the glaucoma model than in control animals.

Presence and Release of ATP from the Retina in an Alzheimer's Disease Model

The aim of this study was to assess the changes of extracellular ATP levels during the progress of Alzheimers disease by using a murine model of the disease. Retinal nucleotide release was measured from flattened whole-mounts stimulated with 59 mM KCl or non-stimulated maintained in Ringer solution. Mice exhibited an increase in retinal ATP release as long as the pathology progressed up to 14 months. This value decreased to normal values by 18 months of age. Changes occurred also when comparing to non-pathological mice. The increase in the presence of ATP levels may contribute, together with other factors, to the changes in the functionality of the retina and the concomitant death of retinal cells.

Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells.

Tear hyperosmolarity is a key event in dry eye. In this work, we analyzed whether hyperosmolar challenge induces ATP release on the ocular surface. Moreover, as extracellular ATP can activate P2X7 receptor, the changes in P2X7 protein levels and its involvement in pathological process triggered by hypertonic treatment were also examined. High-performance liquid chromatography analysis revealed that ATP levels significantly increased in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge as well as in dry eye patients as compared to control subjects. A significant reduction in cell viability was detected after hyperosmolar treatment, indicating that the rise in ATP release was mainly due to cell lysis/death. Additionally, vesicular nucleotide transporter was identified in both cell lines and their protein expression was upregulated in hypertonic media. P2X7 receptor truncated form together with the full-length form was identified in both cell lines, and experiments using specific antagonist and agonist for P2X7 indicated that this receptor did not mediate cell death induced by hyperosmolar stress. In conclusion, hyperosmotic stress induces ATP release. Extracellular ATP can activate P2X7 receptor leading to cytotoxicity in many cells/tissues; however, this does not occur in human corneal and conjunctival epithelial cells. In these cells, the presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior on the ocular surface.

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.

Lactoferrin Levels in Tears are Increased by the Topical Application of Diadenosine Tetraphosphate.

ABSTRACT Purpose: This study was undertaken to determine the effect of the topical application of diadenosine tetraphosphate on lactoferrin levels in rabbit tears. Methods: Diadenosine tetraphosphate was topically instilled in a single-dose, tear samples were collected by micropipette and lactoferrin was measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results: The concentration of lactoferrin in rabbit tears was significantly increased 1 h after diadenosine tetraphosphate application, remaining elevated for 3 h more. This effect was blocked by P2 receptors antagonists. Conclusions: Topical application of diadenosine tetraphosphate stimulates the secretion of lactoferrin in rabbit tears through P2 receptor activation.

Beta2 adrenergic receptor silencing change intraocular pressure in New Zealand rabbits

Purpose/aim Glaucoma consists of a group of progressive optic neuropathies that are characterized by degeneration of the optic nerve and irreversible visual filed loss. Elevated intraocular pressure is the only proven treatable risk factor and commercial products used for glaucoma treatment are focused in lowering intraocular pressure. These drugs can have various undesirable side effects and this invites to look for new strategies. The purpose of this work is to study the use of a siRNA (small interfering RNA) to selectively silence beta2 adrenergic receptors and to see whether it reduces IOP (intraocular pressure). Material and methods Topical instillation of beta2 adrenergic receptors small-interfering RNA (siRNA, 25–250 μg) was applied and IOP was measured with a Tonopen XL up to 9 consecutive days. The effect of such siRNA was compared to commercial compounds such as Timoftlol, Trusopt and Xalatan, and it was also analyzed if some anatomical changes occurred by microscopy. Results siRNA designed for beta2 adrenergic receptor induced a reduction of intraocular pressure (IOP) of 30 ± 5%, compared to a control (scrambled siRNA). The results in terms of IOP decrease were similar to that found with commercial compounds but a long-lasting hypotensive action was shown by beta2 adrenergic receptor siRNA treatment as compared to commercial drugs. No apparent side effects were observed in the ocular structures. Conclusion The use of siRNA against the beta2 adrenergic receptors could provide an interesting therapeutic strategy for glaucoma treatment.

Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation

ABSTRACT Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N‐acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non‐pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N‐acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin‐dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N‐acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca‐calmodulin and the calmodulin‐dependent protein kinase II are involved. HIGHLIGHTSTRPV4 activation promotes the synthesis of melatonin.This synthesis is mediated by the enzyme AANAT phosphorylation.AANAT phosphorylation is carried out by Calcium Calmodulin Dependent Kinase II.The increase of melatonin triggered by TRPV4 permits the control of IOP.