Catabolism of Ap4A and Ap5A by rat brain synaptosomes

Adenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5',5'''-P1,P5-pentaphosphate (Ap5A) are stored in and released from rat brain synaptic terminals. In the present study we investigated the hydrolysis of dinucleotides (Ap4A and Ap5A) in synaptosomes from the cerebral cortex of adult rats. Ap4A and Ap5A, but not Ap3A, were hydrolyzed at pH 7.5 in the presence of 20 mM Tris/HCl, 2.0 mM MgCl2, 10 mM glucose and 225 mM sucrose at 37oC. The disappearance of the substrates measured by FPLC on a mono-Q HR column was both time and protein dependent. Since synaptosome integrity was at least 90% at the end of the assay, hydrolysis probably occurred by the action of an ecto-enzyme. Extracellular actions of adenine dinucleotides at central nervous system terminate due to the existence of ecto-nucleotidases which specifically cleave these dinucleotides. These enzymes in association with an ATP diphosphohydrolase and a 5'-nucleotidase are able to promote the complete hydrolysis of dinucleotides to adenosine in the synaptic cleft.

Purinergic receptors in ocular inflammation

Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly 'tuned,' can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P(1),P(4)-diadenosine tetraphosphate (Ap4A), and P(1),P(5)-diadenosine pentaphosphate (Ap5A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particular A2A adenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A3 receptor, selective agonists like N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Papel biológico del diadenosin tetrafosfato en el ojo: efecto sobre la composición lacrimal e implicación en la función de barrera corneal

El Ap4A es una molécula con un amplio papel biológico en el ojo. Se ha descrito su implicación en procesos de secreción lagrimal, cicatrización epitelial, regulación de la presión intraocular, y presenta también un papel neuroprotector sobre los terminales simpáticos que inervan el cuerpo ciliar. El objetivo de este trabajo ha sido analizar la participación del Ap4A en otras posibles funciones a nivel ocular. En concreto se ha estudiado la capacidad de este dinucléotido para estimular la liberación de proteínas lagrimales de acción antibacteriana tales como la lisozima y la lactoferrina. Por otra parte se ha investigado su efecto como modulador de la función de barrera de la córnea a través de la regulación de los niveles de expresión de proteínas constituyentes de las tight junctions (TJ). Dicho efecto sobre la barrera puede tener una importante repercusión en la entrada de fármacos y en la consiguiente eficacia terapéutica de los mismos. Por último, se ha estudiado la posible implicación del dinucleótido en el proceso de edematización descrito en modelos animales glaucomatosos tales como el ratón DBA/2J. Con el objetivo de averiguar si la activación de Ap4A inducía un efecto sobre la producción de dos proteínas antimicrobianas relevantes de la lágrima (lisozima y lactoferrina) se realizaron ensayos en la lágrima de conejos albinos de Nueva Zelanda, mediante las técnicas de agar-agar y ELISA. Los resultados obtenidos demostraron que Ap4A produce un aumento en la concentración de lisozima y de lactoferrina del 93% y 24%, respectivamente, frente a valores basales, y este efecto está mediado por receptores de tipo P2...

Mecanismos celulares activados por receptores P2Y₂ en los procesos ciliares: implicaciones en la hipertensión ocular y el glaucoma

El glaucoma es una neuropatía óptica crónica de etiología multifactorial, que provoca una pérdida progresiva e irreversible de la visión debida a la degeneración de los axones del nervio óptico y la muerte apoptótica de las células ganglionares de la retina (Downs et al., 2011; Quigley, 2011; Soto et al., 2014). Actualmente es la principal causa de ceguera irreversible en el mundo (Pascolini et al., 2012). Aunque su etiología aún no está clara, se sabe que el principal factor de riesgo para desarrollar la enfermedad es el aumento de la PIO debido a anomalías en la dinámica del humor acuoso (Casson et al., 2012). La presencia de receptores purinérgicos P2X y P2Y ha sido descrita en las principales estructuras oculares relacionadas con la producción y drenaje del humor acuoso, los procesos ciliares y la malla trabecular, que son bañadas por dicho fluido (Guzman-­‐Aranguez et al., 2013). Al mismo tiempo, se ha referido la presencia del dinucleótido Ap4A, agonista de dichos receptores, en el humor acuoso, y cómo sus niveles se veían significativamente incrementados en pacientes glaucomatosos (Castany et al., 2011). El objetivo de esta tesis ha sido evaluar la implicación de los receptores purinérgicos P2Y2 de los procesos ciliares en la hipertensión ocular. Más concretamente determinar el papel del Ap4A a través de la activación de dichos receptores sobre la movilización del ion cloruro y del agua a través de las proteínas acuaporinas (AQPs), principales componentes del humor acuoso. Por otra parte, se ha investigado el origen de los niveles incrementados de Ap4A debidos al aumento de la PIO, caracterizando uno de los posibles sensores celulares implicados en dicho proceso. Al mismo tiempo se han evaluado y caracterizado los diferentes mecanismos implicados en dicha liberación...

Diadenosine polyphosphates in the tears of aniridia patients

Purpose To quantify diadenosine polyphosphate levels in tears of congenital aniridia patients to estimate the ocular surface changes associated with congenital aniridia compared to normal individuals. Methods Fifteen patients diagnosed with congenital aniridia and a control group of forty volunteers were studied. Tears were collected to quantify the levels of diadenosine polyphosphates Ap4A and Ap5A by high-performance liquid chromatography (H.P.L.C). Break-up time (BUT), corneal staining, McMonnies questionnaire and the Schirmer I test were applied to both groups. Results Dinucleotides in congenital aniridia patients were higher than in control subjects. For the congenital aniridia group, under 15 years old, the values were 0.77 ± 0.01 μm and 0.17 ± 0.02 μm for Ap4A and Ap5A, respectively. The group aged from 15 to 40 years old provided concentrations of 4.37 ± 0.97 μm and 0.46 ± 0.05 μm for Ap4A and Ap5A, the group over 40 gave concentrations of 11.17 ± 5.53 μm and 0.68 ± 0.17 μm for Ap4A and Ap5A. Dinucleotide concentrations increased with age, being statistically significant different among the three age groups (p < 0.05). Congenital aniridia patients showed a normal tear secretion and no dry eye McMonnies scores, except for the group over 40 years old. BUT values decreased and corneal staining increased with age and correlated with the levels of diadenosine polyphosphates (p < 0.05). Conclusions The levels of dinucleotides in tears increase in aniridia patients compared with healthy subjects, and they seem to be related with the progression of corneal disorders in aniridia patients, both of which increase with ageing.

Diadenosine polyphosphates after laser in situ keratomileusis and photorefractive keratectomy refractive techniques

Purpose:  To study the concentrations of diadenosine polyphosphates in the ocular surface after PRK and LASIK. Methods:  Sixty-one patients (30 males and 31 females) with ages ranging from 20 to 63 (34.04 ± 9.13 years) were recruited in Balear Institute of Ophthalmology, Palma de Mallorca, Spain. LASIK was performed in 92 eyes of 46 patients and PRK in 25 eyes of 15 patients. Variations in the levels of diadenosine polyphosphate (Ap4A and Ap5A), Schirmer I (Jones test), TBUT, corneal staining together with the Dry Eye Questionnaire to evaluate discomfort and dryness were studied. All tests were performed at the preoperative visit and at 1-day, 2-week, 1-month and 3-month postoperative visits. Results:  Ap4A showed a 5 and 3.5 fold increase at the 1-day visit for LASIK and PRK, respectively. LASIK patients continued having higher statistically significant concentrations (p = 0.01) all over the follow-up. Ap5A showed no significant differences at any visit. Tear volume decreased during the 3 months in LASIK. The PRK cases had a normal volume at 1 month. TBUT in LASIK increased at the 1-day visit (p = 0,002) and decreased from the 2 weeks onwards and for the PRK, decreased by a 35% at the 1-day visit and kept reduced for a month. Discomfort only increased at the 1-day visit (p = 0.007). Dryness frequency was similar in all visits. Conclusions:  Ap4A levels only are increased in refractive surgery patients during the first day after the surgery. This increasing suggests that Ap4A may help accelerating the healing process.

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 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.

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

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.

Corneal Re-epithelialization Stimulated by Diadenosine Polyphosphates Recruits RhoA/ROCK and ERK1/2 Pathways

Purpose. To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap4A and Ap3A. Methods. In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap4A or Ap3A 100 μM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 μM, U0126 100 μM, Y27632 100 nM, and (−)-blebbistatin 10 μM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap4A and Ap3A (100 μM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap4A or Ap3A (100 μM) with U0126 and Y27632 (100 nM). Results. In the presence of Ap4A, U0126, Y27632, AG1478, and (−)-blebbistatin, reduced the migration rate compared to the effect of Ap4A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap4A, respectively). In the presence of Ap3A 100 μM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap3A alone, whereas AG1478 and (−)-blebbistatin (P < 0.0001 versus Ap3A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap4A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. Conclusions. The activation of the Ap4A/P2Y2 receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap3A/P2Y6 receptor signals only the MAPK pathway.

Diadenosine Polyphosphates in Tears of Sjögren Syndrome Patients

Purpose.: To analyze the levels of diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) in tears of subjects with Sjögren syndrome and to compare them with those in a control group. Methods.: Twelve subjects with a diagnosis of Sjögren syndrome and 20 healthy control subjects were invited to participate in the present study. Schirmer strips were used to measure tear secretion (Schirmer I test) and to collect tears. Ap4A and Ap5A were measured by high-pressure liquid chromatography (HPLC), and a dry eye questionnaire (DEQ) was used to evaluate dry eye symptomatology. Results.: The mean concentrations of Ap4A and Ap5A in the Sjögren syndrome group were 2.54 ± 1.02 and 26.13 ± 6.95 μM, respectively. This group of patients was divided in two subgroups: four patients with normal tear production and eight patients with low tear production. Concentrations of Ap4A, and Ap5A in patients with normal tear production (Schirmer test result, 12.3 ± 1.2 mm) were 0.47 ± 0.20 and 8.03 ± 3.27 μM, respectively. In the patients with low tear production (Schirmer test result, 1.0 ± 0.3 mm), the concentrations were 4.09 ± 1.36 and 39.51 ± 8.46 μM, respectively and in the control group, 0.13 ± 0.03 and 0.04 ± 0.02 μM, respectively. Conclusions.: Patients with Sjögren syndrome have abnormally elevated concentrations of diadenosine polyphosphates, indicating that these compounds could be used in the diagnosis of this disease.

Dual Roles of Diadenosine Polyphosphates in Corneal Epithelial Cell Migration

Purpose. To investigate the influence of diadenosine polyphosphates on the rate of corneal epithelial cell migration. Methods. Primary corneal epithelial cell cultures were obtained from New Zealand White rabbits. Immunocytochemical experiments were performed by fixing the cells with 4% paraformaldehyde (PFA) and incubated with cytokeratin 3 primary antibody, which was subsequently incubated with a secondary IgG mouse labeled with FITC, and the cells were observed under confocal microscopy. Migration studies were performed by taking confluent monolayers that were wounded with a pipette tip and challenged with different di- and mononucleotides with or without P2 antagonist (n = 8 each treatment). For concentration–response analysis, compounds were tested in doses ranging from 10−8 to 10−3 M (n = 8). The stability of the dinucleotides was assayed by HPLC, with an isocratic method (n = 4). Results. Cells under study were verified as corneal epithelial cells via the immunocytochemical analysis. Cell migration experiments showed that Ap4A, UTP, and ATP accelerated the rate of healing (5, 2.75, and 3 hours, respectively; P < 0.05; P < 0.001), whereas Ap3A, Ap5A, and UDP delayed it (6.5, 10, and 2 hours, respectively; P < 0.05). ADP did not modify the rate of migration. Antagonists demonstrated that Ap4A and Ap3A did activate different P2Y receptors mediating corneal wound-healing acceleration and delay. Concerning the possible degradation of the dinucleotides, it was almost impossible to detect any products resulting from their cleavage. Conclusions. Based on the pharmacological profile of all the compounds tested, the two main P2Y receptors that exist in these corneal cells are a P2Y2 receptor accelerating the rate of healing and a P2Y6 receptor that delays this process.

In vitro and in vivo delivery of the secretagogue diadenosine tetraphosphate from conventional and silicone hydrogel soft contact lenses

Purpose To evaluate the possible use of soft contact lenses (CL) to improve the secretagogue role of diadenosine tetraphosphate (Ap4A) promoting tear secretion. Methods Two conventional hydrogel CL (Omafilcon A and Ocufilcon D) and two silicone hydrogel (SiH) CL (Comfilcon A and Balafilcon A) were used. Ap4A was loaded into the lenses by soaking in a 1 mM Ap4A solution during 12 h. In vitro experiments were performed by placing the lenses in multi-wells during 2 h containing 1 ml of ultrapure water. 100 μl aliquots were taken at time zero and every minute for the first 10 min, and then every 15 min. In vivo experiments were performed in New Zealand rabbits and both the dinucleotide release from SiH and tear secretion were measured by means of Schirmer strips and high-pressure liquid chromatography (HPLC) analysis. Results Ap4A in vitro release experiments in hydrogel CL presented a release time 50 (RT50) of 3.9 ± 0.2 min and 3.1 ± 0.1 min for the non-ionic and the ionic CL, respectively. SiH CL released also Ap4A with RT50 values of 5.1 ± 0.1 min for the non-ionic and 2.7 ± 0.1 min for the ionic CL. In vivo experiments with SiH CL showed RT50 values of 9.3 ± 0.2 min and 8.5 ± 0.2 min for the non-ionic and the ionic respectively. The non-ionic lens Ap4A release was able to induce tear secretion above baseline tear levels for almost 360 min. Conclusion The delivery of Ap4A is slower and the effect lasts longer with non-ionic lenses than ionic lenses.

Increased Levels of Diadenosine Polyphosphates in Dry Eye

Purpose. To analyze the levels of the diadenosine polyphosphates Ap4A and Ap5A in tears, in a set of control subjects and in groups of symptomatic and nonsymptomatic persons with dry eye. Methods. Ninety-seven subjects participated in the study. The subjects were divided into five experimental groups: control subjects; symptomatic patients with normal tear secretion; symptomatic patients with low tear secretion; forced blink; and corneal mechanical stimulation provided by a gas esthesiometer. The Schirmer I test was used to measure and collect tear secretions from each subject. All samples were processed by high pressure liquid chromatography (HPLC) and their Ap4A and Ap5A levels determined. Results. The levels of Ap4A and Ap5A in tears were greater in all symptomatic patients than in control subjects, especially in symptomatic subjects with low tear secretion. Within the symptomatic subjects with normal tear secretion, significant differences in concentrations of Ap4A and Ap5A were found between men and women. In the forced blink experiments, concentrations of the Ap4A and Ap5A rose with increasing blink frequency. When the cornea was mechanically stimulated, the levels of Ap4A and Ap5A rose significantly during both moderate and high-flow rate tests. Conclusions. The increased levels of Ap4A and Ap5A in tears of patients with dry eye allow these dinucleotides to be used as objective biomarkers in dry eye conditions.

Changes in Diadenosine Polyphosphates during Alignment-Fit and Orthokeratology Rigid Gas Permeable

Purpose.: To evaluate the levels of dinucleotides diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) in tears of patients wearing rigid gas permeable (RGP) contact lenses on a daily wear basis and of patients wearing reverse-geometry RGP lenses overnight for orthokeratology treatment. Methods.: Twenty-two young volunteers (10 females, 12 males; 23.47 ± 4.49 years) were fitted with an alignment-fit RGP lens (paflufocon B) for a month, and after a 15-day washout period they were fitted with reverse-geometry RGP lenses for corneal reshaping (paflufocon D) for another month. During each period, tears were collected at baseline day 1, 7, 15, and 28. Ap4A and Ap5A were measured by high-pressure liquid chromatography (HPLC). Additionally, corneal staining, break-up time (BUT), Schirmer test, and dryness symptoms were evaluated. Results.: Ap4A concentrations increased significantly from baseline during the whole period of daily wear of RGP lenses (P < 0.001); concentration was also significantly higher than in the orthokeratology group, which remained at baseline levels during the study period except at day 1 (P < 0.001) and day 28 (P = 0.041). While BUT and Schirmer remained unchanged in both groups, discomfort and dryness were significantly increased during alignment-fit RGP daily wear but not during the orthokeratology period. Conclusions.: Daily wear of RGP lenses increased the levels of Ap4A due to mechanical stimulation by blinking of the corneal epithelium, and this is associated with discomfort. Also, orthokeratology did not produce symptoms or signs of ocular dryness, which could be a potential advantage over soft contact lenses in terms of contact lens-induced dryness.