Person: Crooke Álvarez, Almudena
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First Name
Almudena
Last Name
Crooke Álvarez
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Óptica y Optometría
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
10 results
Search Results
Now showing 1 - 10 of 10
Item Melatonin Receptors Trigger cAMP Production and Inhibit Chloride Movements in Nonpigmented Ciliary Epithelial Cells(Journal of Pharmacology and Experimental Therapeutics, 2015) Huete Toral, Fernando; Crooke Álvarez, Almudena; Martínez Águila, Alejandro; Pintor Just, Jesús JerónimoMelatonin and its analog 5-MCA-NAT (5-methylcarboxyamino-N-acetyl tryptamine) are active compounds reducing intraocular pressure (IOP). This action is mediated through MT2 and the putative MT3 melatonin receptor, producing a transient reduction of IOP that lasts for a few hours and has not yet been characterized. The use of melatonin and its analog are causing a decrease in chloride efflux from rabbit nonpigmented epithelial cells (NPE), possibly explaining the decrease in IOP. Melatonin and 5-MCA-NAT inhibited rabbit NPE chloride release in a concentration-dependent manner, whereas the pD2 values were between 4.5 ± 1.2 and 4.4 ± 1.0, respectively. Melatonin hypotensive action was enhanced by the presence of MT2 antagonists, such as DH97 (N-pentanoyl-2-benzyltryptamine) and 4-P-P-DOT (4-phenyl-2-propionamidotetralin) and by the nonselective melatonin receptor antagonist luzindole. Prazosin (1.5 µM) partially reverses the melatonin action by acting as a selective MT3 antagonist. However, at 15 nM it acts as an α-adrenergic receptor antagonist, enhancing the melatonin effect. Regarding the intracellular pathways triggered by melatonin receptors, neither phospholipase C/protein kinase C pathway nor the canonical reduction of intracellular cAMP was responsible for melatonin or 5-MCA-NAT actions. On the contrary, the application of these substances produced a concentration-dependent increase of cAMP, with pD2 values of 4.6 ± 0.2 and 4.9 ± 0.7 for melatonin and 5-MCA-NAT, respectively. In summary, melatonin reduces the release of chloride concomitantly to cAMP generation. The reduction of Cl− secretion accounts for a decrease in the water outflow and therefore a decrease in aqueous humor production. This could be one of the main mechanisms responsible for the reduction of IOP after application of melatonin and 5-MCA-NAT.Item The role and therapeutic potential of melatonin in age-related ocular diseases(Journal of Pineal Research, 2017) Crooke Álvarez, Almudena; Huete Toral, Fernando; Colligris, Basilio; Pintor Just, Jesús JerónimoThe eye is continuously exposed to solar UV radiation and pollutants, making it prone to oxidative attacks. In fact, oxidative damage is a major cause of age-related ocular diseases including cataract, glaucoma, age-related macular degeneration and diabetic retinopathy. Since the nature of lens cells, trabecular meshwork cells, retinal ganglion cells, retinal pigment epithelial cells and photoreceptors is post-mitotic, autophagy plays a critical role in their cellular homeostasis. In age-related ocular diseases, this process is impaired, thus, oxidative damage becomes irreversible. Other conditions such as low- grade chronic inflammation and angiogenesis also contribute to the development of retinal diseases (glaucoma, age-related macular degeneration and diabetic retinopathy). As melatonin is known to have remarkable qualities such as antioxidant/antinitridergic, mitochondrial protector, autophagy modulator, anti-inflammatory and anti-angiogenic, it can represent a powerful tool to counteract all these diseases. The present review analyzes the role and therapeutic potential of melatonin in age-related ocular diseases, focusing on nitro-oxidative stress, autophagy, inflammation and angiogenesis mechanisms.Item Signs and Symptoms of Dry Eye in Keratoconus Patients: A Pilot Study(Current Eye Research, 2015) Carracedo Rodríguez, Juan Gonzalo; Recchioni, Alberto; Alejandre Alba, Nicolás; Martín Gil, Alba; Crooke Álvarez, Almudena; Jiménez Alfaro-Morote, Ignacio; Pintor Just, Jesús JerónimoPurpose: 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.Item Low expression of CD39 and CD73 genes in centenarians compared with octogenarians(Immunity & Ageing, 2017) Crooke Álvarez, Almudena; Martínez Henández, Juan; Martínez López, Joaquín; Cruz Jentoft, Alfonso; Huete Toral, Fernando; Pintor Just, Jesús JerónimoAgeing involves a progressive decline of the body’s regulatory systems including immune system. Adenosine regulates immune function by interaction with its receptors, mainly adenosine A2A receptor, present on the surface of immune cells. Furthermore, cellular response to this nucleoside is highly dependent on its extracellular concentration that is regulated by ecto-enzymes such as CD39 and CD73. Therefore, the aim of this study was to investigate the effect of age on adenosine A2A receptor, CD39 and CD73 gene expression. Changes in mRNA were measured by quantitative PCR from peripheral blood of young, middle-aged and older adults as well as centenarians. Centenarians showed a prominent decrease of CD39 and CD73 mRNA in comparison with older adults. Regarding to adenosine A2A receptor, we detected two subgroups of centenarians with high and low level of transcript. Additionally, adenosine A2A receptor mRNA level of centenarians, did not correlate with their cognitive impairment. In summary, our pilot study suggests that unlike of adenosine A2A receptor, the level of CD39 as well as CD73 mRNA could be a hallmark of successful human ageing.Item An update on dry eye disease molecular treatment: Advances in drug pipelines(Expert Opinion on Pharmacotherapy, 2014) Colligris, Basilio; Crooke Álvarez, Almudena; Huete Toral, Fernando; Pintor Just, Jesús JerónimoIntroduction: Dry eye disease is a common disorder provoking changes in tear film and ocular surface. Untreated dry eye could cause ocular infections, corneal ulcer and blindness. Only a few drugs are authorized so far for the treatment of dry eye disease and the possibilities of evolution in this sector are immense. Consequently, a significant number of new potential solutions are under development or placed in the pharmaceutical pipeline, promising better results and lesser side effects. Areas covered: In this article, the corresponding literature and recent Phase III clinical trial data and the corresponding literature, for dry eye disease treatment are reviewed, revealing the new strategic movements in drug pipelines. Expert opinion: From the clinical trial results, the advancement in tear substitutes and secretagogues in addressing specific deficiencies of tear components even though not resolving the underlying conditions of the disease is evident. The vast majority of new compounds under development are anti-inflammatories, steroids, non-steroids and antibiotics; however, there are also some novel lubricating drops and mucin-tear secretagogues. A future aggressive therapy for dry eye, depending on the severity of the symptoms, would include combinations of soft steroids, anti-inflammatories, such as cyclosporine A, with the addition of the new polyvalent mucin and tear secretagogues.Item The role of dinucleoside polyphosphates on the ocular surface and other eye structures(Progress in Retinal and Eye Research, 2016) Carracedo Rodríguez, Juan Gonzalo; Crooke Álvarez, Almudena; Guzmán Aránguez, Ana Isabel; Pintor Just, Jesús JerónimoDinucleoside 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 patient's 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.Item Effect of Melatonin and Analogues on Corneal Wound Healing: Involvement of Mt2 Melatonin Receptor(Current Eye Research, 2015) Crooke Álvarez, Almudena; Guzmán Aránguez, Ana Isabel; Mediero Muñoz, Aránzazu; Alarma Estrany, Pilar; Carracedo Rodríguez, Juan Gonzalo; Peláez García, María Teresa; Peral Cerda, María Asunción; Pintor Just, Jesús JerónimoPurpose: We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial wound healing. Methods: New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discs soaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameter was measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatonin antagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and then every 6 hours together with melatonin. To confirm the presence of MT2 receptors in corneal epithelial cells immunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelial cells were performed. The tear components were extracted then processed by HPLC to quantify melatonin in tears. Results: Migration assays revealed that melatonin and particularly the treatment with the MT2 agonist IIK7, accelerated the rate of healing (p < 0.001). The application of the non-selective melatonin receptor antagonist luzindole and the MT2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on wound healing (both p < 0.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT2 melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determined its daily variations. Conclusions: These data suggest that MT2 receptors are implicated in the effect of melatonin on corneal wound healing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhance epithelial wound healing in ocular surface disease.Item Effect of Melatonin and Its Analogs on Tear Secretion(The Journal of pharmacology and experimental therapeutics, 2019) Navarro Gil, Francisco Javier; Huete Toral, Fernando; Crooke Álvarez, Almudena; Domínguez Godínez, Carmen Olalla; Carracedo Rodríguez, Juan Gonzalo; Pintor Just, Jesús JerónimoMelatonin has been shown to enhance tear secretion associated with dinucleotide diadenosine tetraphosphate. This study investigated the isolated action of melatonin and its analogs, agomelatine, N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl) ethanamine (IIK7), and 5-methoxycarbonylamino-N-cetyltryptamine (5-MCA-NAT) (10 µl at 100 µM), on tear secretion when applied topically in the rabbit cornea and its relationship with the melatonin MT1, MT2, and MT3/quinone reductase QR2 receptors. The results showed a significant increase in tear secretion, with a maximal effect at 60 minutes for the agonists (138.9% ± 6.5%, 128.9% ± 6.4%, and 120.0% ± 5.2%, respectively; P < 0.05; 100% control) but not for melatonin (101.6% ± 7.9%; P > 0.05). Agonist action was tested combined with the antagonists DH97 (MT2 selective), prazosin (MT3/QR2 inhibitor), and luzindole (nonselective MT membrane receptor) (10 µl at 100 µM). DH97 reversed the effect of agomelatine, IIK7, and 5-MCA-NAT up to 30.85% ± 7.6%,108% ± 7.2%, and 87.01% ± 7.6%, respectively (P < 0.05; 100% control). Luzindole antagonized agomelatine and 5-MCA-NAT up to 67.35% ± 7.6% and 92.12% ± 8%, respectively (P < 0.05). Prazosin only reversed 5-MCA-NAT action up to 84.2% ± 7.7% (P < 0.05). These results suggest different pathways for the agonists to act through MT membrane receptors. Therefore, agomelatine, IIK7, and 5-MCA-NAT act through MT membrane receptors as secretagogues of tear secretion, and these analogs could be considered excellent therapeutic candidates for dry eye treatment.Item Silencing of P2Y2 receptors reduces intraocular pressure in New Zealand rabbits(British Journal of Pharmacology, 2012) María Jesús Perez de Lara; Concepción Santano; Martín Gil, Alba; Crooke Álvarez, Almudena; Peral Cerda, María Asunción; Pintor Just, Jesús JerónimoBACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: Agonists of the P2Y2 receptor a as well as P2 antagonists were applied to eyes of New Zealand rabbits, and the changes in IOP were followed for up to 6 h. Cloning of the P2Y2 receptor cDNA was done using a combination of degenerate reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). siRNA was synthesized and tested by immunohistochemistry. KEY RESULTS: Single doses of 2-thioUTP, UTP-γ-S and UTP increased IOP. This behaviour was concentration-dependent and partially antagonized by reactive blue 2. Silencing the P2Y2 receptor was observed in the ciliary body by immunohistochemistry labelling, where a reduction in the immunofluorescence was observed. This reduction in the expression of the P2Y2 receptor was concomitant with a reduction in IOP, which was measurable 24 h after treatment with the siRNA, maximal after 2 days, followed by a slow increase towards control values for the following 5 days. Application of the P2Y2 agonists after pretreatment of the animals with this siRNA did not produce any change in IOP. CONCLUSIONS AND IMPLICATIONS: P2Y2 receptors increase IOP in New Zealand rabbits. The application of a siRNA for this receptor significantly reduced IOP, suggesting that this technology might be used for the treatment of glaucoma.Item Involvement of carbonic anhydrases in the ocular hypotensive effect of melatonin analogue 5‐MCA‐NAT(Journal of Pineal Research, 2011) Martínez Águila, Alejandro; Crooke Álvarez, Almudena; Huete Toral, Fernando; Martín Gil, Alba; Pintor Just, Jesús JerónimoWe 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.