Beta2 adrenergic receptor silencing change intraocular pressure in New Zealand rabbits / Cambio de la presión intraocular en conejos de Nueva Zelanda mediante silenciamiento del receptor adrenérgico Beta2

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 g) 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

Objetivo: El glaucoma consiste en un grupo de neuropatías ópticas progresivas caracterizadas por degeneración del nervio óptico y pérdida irreversible del campo visual. La elevación de la presión intraocular es el único factor de riesgo tratable probado, centrándose los productos comerciales para el tratamiento del glaucoma en la reducción de la presión intraocular. Estos fármacos pueden tener diversos efectos secundarios indeseados, lo cual invita a buscar nuevas estrategias. El objetivo de este trabajo es estudiar el uso de un ARNip (ARN pequeño de interferencia) para silenciar selectivamente los receptores adrenérgicos beta2, y comprobar si reduce la PIO (presión intraocular). Material y métodos: Se realizó instilación tópica de ARN pequeño de interferencia para los receptores adrenérgicos beta2 (ARNip, 25-250 g), midiéndose la PIO con Tonopen XL hasta nueve días consecutivos. Se comparó el efecto de dicho ARNip con componentes comerciales tales como Timoftlol, Trusopt y Xalatan, analizándose asimismo mediante microscopio si se producía cualquier cambio anatómico. Resultados: ARNip diseñado para el receptor adrenérgico beta2 indujo una reducción de la presión intraocular (PIO) de 30 ± 5%, en comparación al control (ARNip de secuencia aleatoria). Los resultados en términos de reducción de la PIO fueron similares a los encontrados utilizando componentes comerciales, aunque el tratamiento con ARNip para el receptor adrenérgico beta2 reflejó una acción hipotensora de larga duración en comparación con los fármacos comerciales. No se observaron efectos secundarios aparentes en las estructuras oculares. Conclusión: El uso de ARNip contra los receptores adrenérgicos beta2 podría aportar una estrategia terapéutica interesante para el tratamiento del glaucoma

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, 25-250µg) 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.

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

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.

Diadenosine tetraphosphate improves adrenergic anti-glaucomatous drug delivery and efficiency.

The effect of the dinucleotide P(1), P(4)-Di (adenosine-5') tetraphosphate (Ap4A) in improving adrenergic anti-glaucomatous delivery by modifying the tight junction proteins of the corneal epithelium was evaluated. Stratified human corneal epithelial cells (HCLE) were treated with Ap4A (100 µM) for 5 min and TJ protein levels and barrier function were analysed by western blotting and transepithelial electrical resistance (TEER), respectively. Western blot experiments showed a significant reduction at 2 h (45% reduction of ZO-1 and 65% reduction of occludin protein levels) as compared to non-treated (control) cells. Two hours after Ap4A treatment, TEER values were significantly reduced (65% as compared to control levels (p < 0.001)), indicating an increase in corneal barrier permeability. Topical application of Ap4A in New Zealand white rabbits two hours before the instillation of the hypotensor compounds (the α2-adrenergic receptor agonist, brimonidine and the ß-adrenergic receptor antagonist, timolol), improved the delivery of these compounds to the anterior chamber as well as their hypotensive action on the intraocular pressure. The results obtained showed that, when Ap4A was topically applied two hours before the adrenergic compounds, the concentration of brimonidine in the aqueous humour increased from 64.3 ± 5.3 nM to 240.6 ± 8.6 nM and from 58.9 ± 9.2 nM to 183.7 ± 6.8 nM in the case of timolol, which also produces a more profound effect on IOP. Therefore, Ap4A treatment results in a better entrance of adrenergic anti-glaucomatous compounds within the eye and consequently improved therapeutic efficiency by increasing corneal epithelial barrier permeability.

Diadenosine polyphosphates release by human corneal epithelium.

Diadenosine polyphosphates are a type of dinucleotides that have been detected in rabbit and human tears. However, their origin and their mechanism of release have not been fully elucidated. In this work we investigated whether the dinucleotides Ap4A and Ap5A can be released from human corneal epithelia as a consequence of shear stress stimuli. In in vitro experiments, concentrations of Ap4A and Ap5A before mechanical stimulus of stratified human corneal epithelial cells were 3.18 ± 0.43 nM and 0.81 ± 0.13 nM, respectively. After shear stimulation, concentrations significantly increased to 12.01 ± 2.19 nM for Ap4A and 2.83 ± 0.41 nM for Ap5A. No significant differences in lactate dehydrogenase activity were detected between non-stimulated stratified human corneal epithelial cells and cells exposed to mechanical shear-stress, indicating that the rise of dinucleotide levels was not due to cell lysis. In in vivo experiments, individuals subjected to a rise in blinking frequency showed a significant increase of Ap4A (∼25-fold when experiment was performed without anaesthetic and 75-fold with anaesthetic) and Ap5A concentration in tears (∼50-fold when experiment was performed without anaesthetic and 125-fold with anaesthetic). Shear-stress stimuli induces Ap4A and Ap5A release from human corneal epithelium, thus explaining the origin of these relevant compounds for the ocular surface biochemistry and physiology.

Topical application of nucleotides increase lysozyme levels in tears.

The present work studies the effects of topical application of nucleotides on rabbit tear lysozyme levels. Lysozyme values were determined by the diffusion in Agar method described by van Bijsterveld in 1974, and the protein amount was obtained by measuring the inhibitory halos around a Whatman n degrees 1 paper disc of 5 mm in diameter. The tested nucleotides were UTP, Ap(4)A and Up(4)U. These compounds were topically instilled in a single-dose in one eye (with the contralateral eye as a control) and the lysozyme halos were measured along 5 hours. The obtained results showed an increase in the lysozyme concentrations of 67%, 93%, and 119% for UTP, Ap(4)A, and Up(4)U, respectively, over the basal levels of lysozyme. For this reason, we suggest these molecules as a potential treatment for the reinforcement of the tear film barrier against ocular infection.

Melatonin Makes Me Feel Awake! Seven Years of Lab Experience (2000-2007)

La investigación sobre el efecto de la melatonina sobre la presión intraocular(PIO) es revisada desde la perspectiva histórica de nuestro laboratorio. La idea original sobre la melatonina modulando la presión intraocular ha sido mejoradagracias al empleo de nuevos compuestos selectivos para el receptor MT2y especialmente para el MT3. El compuesto selectivo 5-metoxiamino N-acetiltriptamina(5-MCA-NAT) se ha convertido en un compuesto muy atractivo debidoa su capacidad de reducir la PIO hasta un 50%, convirtiéndose en un compuestomuy interesante para el tratamiento de la hipertensión ocular asociada al glaucoma.Se han desarrollado y ensayado más compuestos que nos han permitido tener unpanorama más preciso acerca de como estos receptores controlan el procesofisiológico de la presión intraocular

The research on the effect of melatonin on intraocular pressure (IOP) isreviewed from the hystorical point of view of our laboratory. The original idea ofmelatonin modulating intraocular pressure has been improved by using selectivecompounds for MT2 and specially melatonin MT3 receptors. The selectivecompound 5-methoxyamino N-acetyltryptamine (5-MCA-NAT) has been anattractive compound due to its ability to reduce IOP about 40%, therefore being agood candidate to the treatment of the ocular hypertension linked to glaucoma.More compounds have been developed and tested permitting us to have a moreaccurate panorama of those receptors controlling the relevant process ofintraocular pressure