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CELLULAR AND MOLECULAR

Adenosine Tetraphosphate, Ap₄, a Physiological Regulator of Intraocular Pressure in Normotensive Rabbit Eyes

Departmentos de Bioquimica (J.P., T.P.) and Optica (A.P.), Escuela Universitaria Optica, Universidad Complutense de Madrid, Madrid, Spain

Received August 14, 2003; accepted October 31, 2003.

	Abstract

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Adenosine 5' tetraphosphate, Ap₄, is a natural nucleotide present in many biological systems. This nucleotide has been found as a constituent of the nucleotide pool present in the aqueous humor of New Zealand rabbits. HPLC analysis confirmed its identity and calculated its concentration levels to be 197 ± 21 nM. When applied topically to the rabbit eyes, this mononucleotide produced a reduction in the intraocular pressure, which was dose-dependent. The pD₂ value calculated from the dose-response curve was 7.28 ± 0.47, which is equivalent to 52.48 nM. The time course of such intraocular pressure reduction presented a maximal decrease of IOP to 75.1 ± 2.3% compared with the vehicle control value (100%), and the effect lasted for more than 2 h. Cross-desensitization studies demonstrated that Ap₄ effect was mediated via a P2X receptor in this system. P2 receptor antagonists suramin, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive blue 2 (RB-2) showed that only the latter was able to revert the effect of Ap₄. Antagonists of adrenoceptors and cholinoceptors were able to partially reverse the effect of this nucleotide; this might indicate a connection with the neural mechanisms that control the intraocular pressure.

Ap₄ was identified first from horse muscle (Lieberman, 1955) and also in other tissues such as rabbit muscle and platelets, yeast spores, and rat liver (Small and Cooper, 1966; Lobaton et al., 1975; Van Dyke et al., 1977). This nucleotide is present and releasable from neurochromaffin cells (Winkler and Carmichael, 1982; Gualix et al., 1996), and its synthesis is carried out by a transphosphorylation process involving ATP (Gualix et al., 1996). When stored in the vesicles, it can be released to the extracellular space together with other transmitters. Once at the extracellular space, this nucleotide can activate both ionotropic P2X and metabotropic P2Y receptors. In this sense, the effect of Ap₄ on the vascular system is to reduce blood pressure by means of the activation of a P2Y receptor; however, in stress situations, such as in an hemorrhage, it produces vasoconstriction by the activation of smooth muscle P2X receptors (Lee et al., 1995b; Lewis et al., 2000). Very recently, Ap₄ has been identified in human myocardial tissue. The application of this nucleotide in isolated rat heart can produce vasodilatation (Westhoff et al., 2003).

It is only recently that the nucleotides are considered as active compounds in the eye (Pintor, 1999). Processes in which extracellular nucleotides can modify ocular physiology include tear secretion, intraocular pressure, and retinal fluid reabsorption (Yerxa 2001; Pintor et al., 2002). Mononucleotides, such as ATP and its synthetic analogs, can modulate intraocular pressure (IOP). There is a clear difference in the way they can modify IOP, because those nucleotides with a P2Y agonistic profile tend to increase IOP, whereas P2X agonists reduce it (Pintor and Peral, 2001). Recently, it has been demonstrated that diadenosine polyphosphates can modify IOP. Diadenosine tetraphosphate (Ap₄A), induces a decrease in IOP, whereas the other members of this family produce an increase in the rabbit's intraocular pressure (Pintor et al., 2003).

Nothing is known about the role of the nucleotide Ap₄ in the eye, its presence, or its physiological behavior. Hence, this investigation will look at the presence of Ap₄ in the rabbit's aqueous humor and describe its effect on the intraocular pressure.

	Materials and Methods

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Animals. Twenty-four normotensive New Zealand White rabbits (males, 2–3 kg) were used. The animals were kept in individual cages with free access to food and water, under controlled cycles (12/12-h light/dark). Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and the statement of the Association for Research in Vision and Ophthalmology on the Use of Animals in Ophthalmic and Vision Research.

Aqueous Humor Collection and HPLC Procedures. Aqueous humor was collected from anesthetized rabbits by means of an intracameral injection. These experiments were performed at the same time, 3:00 PM, when IOP has recovered completely from the night period. Briefly, New Zealand White rabbits were anesthetized with 1.5 mg/kg propofol (Abbott Laboratories, Madrid, Spain). Aqueous humor (500 µl) was removed with a syringe connected to a 30-gauge needle in the scero-corneal limbus. Samples were stored at –35°C before treatment through SEP-Pak Accell QMA cartridges (Waters, Milford, MA). Samples were chromatographed through these cartridges, and the elution of these compound was obtained by applying 500 µl of a mixture containing 0.1 N HCl, 0.2 N KCl. Eluates were neutralized with 10 N KOH before HPLC analysis (Rotllán et al., 1991).

The HPLC system consisted of a Waters 1515 Isocratic HPLC pump, a 2487 dual wavelength absorbance detector, and a Reodyne injector, all controlled by the software Breeze from Waters. The column used was a NovaPak C18 (15 cm in length, 0.4 cm in diameter) also from Waters. The mobile phase consisted of 10 mM KH₂PO₄, 2 mM tetrabutyl ammonium, 17% acetonitrile, pH 7.5. Detection was monitored at 260-nm wavelength.

To confirm the chemical nature of the putative peak identified as Ap₄, enzymatic digestion with alkaline phosphatase (EC 3.1.3.1 [EC] ) 0.3 U/ml (molecular biology grade) was used. Incubation with this enzyme and the putative nucleotide was carried out for 10 min, and the digested products were analyzed by HPLC.

Animals used for aqueous humor studies were not used for IOP experiments after 2 weeks of recovery.

Intraocular Pressure Measurements. Intraocular pressure was measured by means of a Tono-Pen XL contact tonometer (Mentor Massachusetts Inc., Norwell, MA). This device has been shown to be the tonometer of choice for measuring intraocular pressures within the range of 3 to 30 mm Hg in rabbits (Abrams et al., 1996). Because IOP changes from the night to day, all the experiments were performed at the same time, 3:00 PM. At this time, IOP remains more stable and permits an objective comparison with vehicle treatment. All measurements fell within this diapson: the mean baseline value of intraocular pressure was 17.0 ± 0.39 mm Hg (n = 100). A topical anesthetic (Colircusí Laboratorios, Cusí, Spain: 0.1 mg · ml^–1 tetracaine plus 0.4 mg · ml^–1 oxybuprocaine in 0.9% saline, diluted 1:3 in 0.9% saline) was applied (10 µl) to the cornea before each measurement of intraocular pressure was made. Ap₄ or saline was applied topically to the cornea in volumes of 10 µl. To compare the effect of Ap₄ with other compounds that reduce IOP, Xalatan (latanoprost 0.005%), 10 µl, was assayed and IOP measured for 6 h.

In a series of experiments, the intraocular pressure was measured twice, 30 min apart, before application of a dose of Ap₄ or saline (0.9% NaCl, vehicle). In establishing dose-effect relationships, measurements of intraocular pressure were made after 30 min and every hour for 6 h after the application. Maximum responses were observed after 2 h. One dose was tested per rabbit per day. In experiments where the ATP P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suramin, and reactive blue 2 (RB-2) were tested, these compounds were applied after the two baseline measurements and 30 min before application of saline or a nucleotide.

Occlusion experiments were carried out in the same manner as with the antagonists. Briefly, Ap₄, ,-meATP, and ATP--S (all 100 µM, 10 µl), were applied 30 min before the administration of Ap₄ at 100 µM (10 µl).

Cholinergic antagonists (hexamethonium and atropine) and noradrenergic antagonists (yohimbine and ICI-118.551), were assayed at a fixed concentration of 10 µg/µl (10 µl instillation), 30 min before the application of the purinergic compounds (Pintor and Peral, 2001).

Drugs. Ap₄, alkaline phosphatase, PPADS, reactive blue 2, and suramin were purchased from Sigma-Aldrich (St. Louis, MO). ,-meATP and ATP--S were purchased from Roche Diagnostics (Mannheim, Germany). Atropine, hexamethonium, ICI-118.551 and yohimbine were from Tocris (Bristol, UK). Xalatan was purchased from Pharmacia Upjohn (Barcelona, Spain). Other reagents were analytical grade from Merck (Darmstadt, Germany).

Analysis of Data. Numerical values are given as mean ± S.E.M. Means of two groups were compared using Student`s t test (unpaired, two-tailed, unless otherwise stated) with a 5% fiducial point of significance, unless otherwise stated.

	Results

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Presence and Quantification of Ap₄ in the Aqueous Humor. The chromatographic analysis of the samples obtained from rabbit's aqueous humor revealed the presence of various peaks that were identified as mono and dinucleotides. In particular, a peak occurred between the corresponding ATP and diadenosine tetraphosphate, Ap₄A peaks. This peak was tentatively identified as Ap₄ compared with a nucleotide standard chromatographed that contained the commercial Ap₄ (Fig. 1). Because the identification by the retention time and comparison with a standard is not enough to verify the presence of this nucleotide in the aqueous humor of the rabbit, the peak was collected and submitted to alkaline phosphatase treatment as described under Materials and Methods. The products obtained after the enzyme digestion, with the presence of ATP, ADP, and AMP, and the concomitant reduction in the peak of the putative Ap₄, indicated that the nucleotide investigated was Ap₄ (Fig. 2). The concentration obtained for this compound in the aqueous humor was 197 ± 21 nM (n = 8), which is smaller than the one obtained for other mononucleotides (Pintor et al., 2002).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Presence of adenosine tetraphosphate in rabbit aqueous humor. Samples analyzed as described under Materials and Methods presented a peak (upper trace) identified tentatively as Ap4, compared with a commercial standard (lower trace, 1000 pmol each). Inset, blowup of the putative adenine tetraphosphate peak. Aqueous humor was taken alway at the same time, 3:00 PM, to avoid the possible effect of circadian rhythms.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Alkaline phosphatase treatment and analysis of putative adenosine tetraphosphate. Ap4 was treated with alkaline phosphatase (molecular biology grade) as described under Materials and Methods. The treatment of the putative Ap4 (top trace) completely changed the chromatographic profile with peaks corresponding to AMP and ADP occurring as digestion products (second trace). The bottom trace represent a standard of 1000 pmol AMP, ADP, and ATP.

Effect of Ap₄ on Intraocular Pressure. Because Ap₄ is present in the rabbit aqueous humor, we wanted to know whether there was any change in the IOP values when this nucleotide was topically applied. Graded doses of Ap₄ ranging from 10^–2 to 10^–11 M were instilled as described previously, to construct a dose-response curve. As it is shown in Fig. 3, Ap₄ was able to reduce intraocular pressure in rabbits in a concentration dependent manner. IOP was reduced to 75.1 ± 2.3% compared with control the values (100%). The analysis of the curve permitted the calculation of the pD₂ value, which was 7.28 ± 0.47, equivalent to an EC₅₀ of 52.48 nM (n = 8).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Dose-response analysis of adenosine tetraphosphate on rabbit intraocular pressure. Effect of instillation of graded doses of Ap4 into the rabbit eye on IOP (solid squares) and the effect of vehicle control (open squares). Each point was obtained 3 h after the application of the nucleotide (maximal effect of Ap4). Initial IOP values were 17 ± 0.39 mm Hg, whereas the maximal effect reduced IOP to 12.6 ± 1.3 mm Hg. All the IOP experiments were started at 3:00 PM to avoid the possible effect of circadian rhythms. Points represented the mean ± S.E.M. (n = 8).

The time course of Ap₄-induced IOP changes was examined. A single dose of 100 µM Ap₄ produced a reduction of IOP, from 17.1 ± 0.6 to 12.5 ± 1.5 mm Hg, which was maximal 2 h after the application of the nucleotide (Fig. 4). The effect was sustained for about 2 to 3 h and returned to normal values within 6 h after (n = 6). To compare the effect of the nucleotide with other substance that is known to reduce IOP, the commercial hypotensive compound Xalatan (latanoprost) was topically applied to the rabbit eyes. Ten microliters of 0.005% Xalatan, produced a marked reduction in normotensive rabbit's IOP, from 16.9 ± 0.8 to 11.3 ± 0.1 mm Hg, as observed in Fig. 4.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Time course of changes in IOP in response to a single instillation of Ap4. A single dose of Ap4 (100 µM,10 µl) produced a decrease in IOP (from 17.1 ± 0.6 to 12.5 ± 1.5 mm Hg) that returned 4 h after the instillation of the nucleotide (solid squares). The IOP hypertensive compound Xalatan (10 µl of 0.005% latanoprost) produced a marked reduction in normotensive rabbit's IOP (from 16.9 ± 0.8 to 11.3 ± 0.1 mm Hg) (open squares). All the IOP experiments were started at 3:00 PM to avoid the possible effect of circadian rhythms. Values represent the mean ± S.E.M. of six independent experiments.

Cross-Desensitization and Studies with P2 Antagonists. To investigate the P2 purinergic receptor involved in the effect of Ap₄, cross-desensitization studies were performed by means of ATP--S and ,-meATP, hypertensive and hypotensive nucleotides, in this model as described previously (Pintor and Peral., 2001).

Homologous cross-desensitization of Ap₄ at a maximal dose of 100 µM, allowed the measurement of the first hypotensive effect but not the one due to the second dose of this nucleotide (n = 6; Fig. 5). A similar behavior was observed when the P2X agonist ,-meATP was applied before Ap₄. When the P2Y agonist ATP--S was applied, it was possible to measure the hypotensive effect of Ap₄, despite the huge hypertensive effect displayed by ATP--S (n = 6; Fig. 5). These experiments suggest the activation of a P2X receptor that is involved in a reduction of IOP as occurs with ,-me ATP and diadenosine tetraphosphate (Pintor and Peral, 2001; Pintor et al., 2003).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Cross-desensitization experiments with different nucleotides. Homologous and heterologous desensitization experiments for Ap4 (at 100 µM, 10 µl). ,-meATP and ATP--S were preincubated as described under Materials and Methods at concentrations of 100 µM (10 µl). All the IOP experiments were started at 3:00 PM to avoid the possible effect of circadian rhythms. Values represent the mean ± S.E.M. of six independent experiments. ***, p < 0.001 versus the effect of ATP--S alone.

Three different P2 antagonists were tested in their ability to reverse the hypotensive effect of Ap₄: suramin, RB-2, and PPADS. As shown in Fig. 6, only RB-2 was able to significantly reverse the effect of Ap₄. Neither PPADS nor suramin could modify the reduction in IOP produced by Ap₄ in a significant way at the single dose tested (n = 6).

View larger version (11K): [in this window] [in a new window]   Fig. 6. Effect of P2 receptor antagonists on Ap4 response. Antagonism of suramin, RB-2, and PPADS (all at 100 µM, 10 µl) on the hypotensive response induced by Ap4 (at 100 µM, 10 µl). All the IOP experiments were started at 3:00 PM to avoid the possible effect of circadian rhythms. Values represent the mean ± S.E.M. of six independent experiments. *, p < 0.05 versus the effect of Ap4 alone.

Studies with Cholinergic and Noradrenergic Antagonists. Intraocular pressure is a process controlled by the nervous system (Bergmanson, 1982). To see whether the effect of Ap₄ is coupled to the nervous system, antagonists of the cholinergic and noradrenergic systems were tested.

The application of the cholinergic antagonists hexamethonium and atropine alone produced a clear increase in the IOP (Fig. 7). When the two cholinergic antagonists were applied 30 min before the instillation of Ap₄, the hypertensive effect of hexamethonium and atropine disappeared, and it was possible to observe a reduction in IOP (due to Ap₄), which was not statistically different from the effect of Ap₄ alone (n = 6; Fig. 7).

View larger version (33K): [in this window] [in a new window]   Fig. 7. Effect of cholinergic and adrenergic receptor antagonists on Ap4 response. The effect of cholinoceptor antagonists (hexamethonium and atropine, 10 µg/µl, 10 µl instillation) and 2 and 2-adrenoceptor antagonists (yohimbine and ICI-118.551, 10 µg/µl, 10-µl instillation) were assayed alone and in the presence of 100 µM Ap4. All the IOP experiments were started at 3:00 PM to avoid the possible effect of circadian rhythms. Values represent the mean ± S.E.M. of six independent experiments. *, p < 0.05 versus the effect of Ap4 alone.

The application of the adrenergic antagonists yohimbine and ICI-118.551 produced also an increase in IOP (Fig. 7). When Ap₄ was applied after the previous instillation of these antagonists, there was a measurable reduction in IOP, nevertheless there were significant differences between this value and the one by Ap₄ alone (n = 6; Fig. 7).

	Discussion

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The nucleotide Ap₄ is a naturally occurring nucleotide present in rabbit aqueous humor as demonstrated by high-performance liquid chromatography. Comparison with a commercial standard, digestion with alkaline phosphatase, and analysis of the digestion products permitted to characterize and identify this compound as a component of the aqueous humor.

Nucleotides and nucleosides have been described in the aqueous humor of different animal models. In this sense, Maul and Sears (1979) described the release of ATP after stimulation of the trigeminal nerve of the rabbit eye as an extracellular source of nucleotides. In the same way, Mitchell et al. (1998) described the release of ATP from bovine ocular ciliary epithelial cells into the aqueous humor. Also, Greiner et al. (1991) showed the presence of phosphate metabolites in ocular humors as well as the presence of the nucleoside adenosine in rabbits (Crosson and Petrovich, 1999). All these works are principally focused on ATP, which has been generally considered as the natural agonist of P2 receptors together with ADP, UTP, and UDP. Nevertheless, other nucleotides such as diadenosine polyphosphates have been described in the rabbit aqueous humor: Ap₄A and diadenosine pentaphosphate (Ap₅A) are present at concentrations in the low micromolar range, and they can exert differential action on IOP (Pintor et al., 2003). In a recent study, a calculation of the concentrations of mononucleotides in the aqueous humor demonstrated that the values obtained for ATP or ADP were about 1 order of magnitude higher than the concentration value obtained for Ap₄ in the present study (Pintor et al., 2003). A lower concentration in the aqueous humor is not indicative that Ap₄ is less relevant than other mononucleotides. For example, in terms of extracellular stability, Ap₄ lasts longer than ATP, as demonstrated by Gomez-Villafuertes et al. (2000). In this way, the rate of hydrolysis of Ap₄ in synaptic terminals is 1.89% per 2 min, whereas ATP hydrolyzes 24.64% for the same period of time. Also, the time course for ATP on IOP in these rabbits is different from the one obtained for Ap₄ (data not shown). ATP effect presents a sharp reduction in IOP, which rapidly returns to normal values continuing toward an hypertensive effects 2 h after the application of the nucleotide (Peral et al., 2001; Pintor et al., 2000b). This biphasic effect is probably due to the gradual degradation of ATP to adenosine, which has been described as an hypertensive compound when acting through adenosine A2 receptors (Crosson and Gray, 1996). There is, therefore, a difference between the effect of ATP and Ap₄ that suggests that Ap₄ remains stable as an active molecule longer than ATP and therefore may act through purinergic receptors.

Ap₄ presents an interesting effect of reducing intraocular pressure in normotensive rabbits. Other adenosine nucleotides have shown a similar behavior. ATP and the analogs ,-meATP and ,-meATP can produce a reduction in the IOP. The reduction in IOP is similar in magnitude to the one produced by Ap₄ (Pintor and Peral, 2001). Moreover, it is probable that in the regulation of IOP by these nucleotides and Ap₄, they are stimulating the same P2X receptor. This conclusion partially supported on the cross-desensitization studies when using ,-meATP and the lack of effect to a second dose of Ap₄. Also because the effect of the hypertensive nucleotide ATP--S (a P2Y agonist in this model) is severely reduced when an application of Ap₄ is used. The blockade by means of the P2 antagonist RB-2 suggests that the action of ,-meATP and Ap₄ are performed through the same P2X receptor. It is interesting to compare the differential effect of the P2 antagonists tested. It would be expected to have a reduction in the effect when suramin is applied, because it is a general P2 receptor antagonist. It needs to be taken into account that all the antagonists have been tested at a single dose and that this does not need to the most effective for all the antagonists. Another possibility is to have differential antagonist permeability. The cornea may be selective to the transport of some of the P2 antagonists but not to others.

Comparison of the pharmacology for the effect of Ap₄ on IOP with other tissues demand a more detailed pharmacological study; nevertheless, some conclusions can be made. For example, Ap₄ has been demonstrated to activate P2X receptors in areas such as smooth muscle and the central nervous system (Lee et al., 1995b; Gomez-Villafuertes et al., 2000). In rat midbrain synaptic terminals, this nucleotide stimulates a heteromeric P2X receptor that present features of a P2X₂/P2X₃ purinergic receptor (Gomez-Villafuertes et al., 2000). This receptor is pharmacologically different from the one described in ocular cells/tissues because in the rat synaptic terminals the nucleotide ATP--S is a full agonist, whereas in the rabbit eye is not. This observation suggests the existence of a P2X receptor in the rabbit eye that presents a subunit composition that does not match with the one described in the rat brain.

Other locations where Ap₄ stimulates P2X receptors are vas deferens, vascular smooth muscle, and mesenteric artery (Bailey and Hourani, 1995b; Lee et al., 1995a; Lewis et al., 2000). In these models, adenosine 5'-tetraphosphate produces vasoconstriction, with this nucleotide being especially active on P2X₁ receptors (Lewis et al., 2000). It is premature to elucidate which subunits form the P2X receptor involved in the reduction of intraocular pressure, according to the preliminary pharmacological studies presented in this work. It is necessary to start with studies at the molecular level with isolated cells from the trabecular meshwork and ciliary processes to fully understand the location and characteristics of this receptor.

An interesting finding is that the levels found in the aqueous humor for Ap₄ are in the range for promoting IOP decrease, as illustrated in the dose-response experiments presented here. A concentration of 197 nM is near to the EC₅₀ value. This suggests that under certain physiological circumstances, the humor levels of Ap₄ can oscillate and therefore they may modulate intraocular pressure. We still do not know what factors influence the possible variations in the Ap₄ concentrations, but if Ap₄ is released from ocular nerve terminals, its concentrations may be regulated, as happens with other neurotransmitters in the eye, such as acetylcholine or epinephrine.

It is also possible that Ap₄ is generated from the hydrolytical cleavage of the dinucleotide Ap₅A. This process, carried out by means of an asymmetrical phosphodiesterase, is relevant extracellularly and could be an alternative to neural release (Zimmermann, 1996; Mateo et al., 1997). Nevertheless this cannot be the only mechanism for Ap₄ production because the concentrations measured of Ap₅A in the aqueous humor were only 80 nM, insufficient to justify the presence of this mononucleotide as a consequence of Ap₅A cleavage if we consider that 100% of Ap₅A is hydrolyzed (Pintor et al., 2003). So far, we do not know which one of these processes is the most likely one or whether there are alternative sites for its release such as the ciliary epithelial cells, which can release adenine nucleotides as reported previously (Mitchell et al., 1998).

Concerning the possible side effects the application of Ap₄ can produce, we focused rabbit's corneal transparency, corneal thickness, and changes in the corneal endothelium (possible polymegathism and changes in cell hexagonality). During the time of the present research, no changes in the parameters measured were observed (data not shown).

In summary, Ap₄ is a mononucleotide present and physiologically active in the rabbit eye. It can modulate intraocular pressure through a mechanism that involves the activation a P2X receptor. This compound may be used for the treatment of those pathologies involved in an abnormal increase in the intraocular pressure.

	Acknowledgements

 
We thank Natasha Hakim for assistance in the preparation of this manuscript.

	Footnotes

 
This work has been supported by a research grant from Inspire Pharmaceuticals, from the Ministry of Science and Technology DGCYT SAF2003-00338 and Fondos Europeos FEDER UCMA-E017.

DOI: 10.1124/jpet.103.058669.

ABBREVIATIONS: Ap₄, adenosine 5'-tetraphosphate; IOP, intraocular pressure; Ap₄A, diadenosine tetraphosphate; ATP--S, adenosine 5'-3-O-thiotriphosphate; ,-me ATP, ,-methylene-adenosine 5' triphosphate; ,-meATP, ,-methylene-adenosine 5' triphosphate; HPLC, high-performance liquid chromatography; PPADS, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid; RB-2, reactive blue 2; Ap₅A, diadenosine pentaphosphate; ICI-118.551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino].

Address correspondence to: Dr. Jesus Pintor, Departmento de Bioquimica y Biologia Molecular IV, E.U. Optica, Universidad Complutense de Madrid, c/Arcos de Jalon s/n, 28037 Madrid, Spain. E-mail: jpintor{at}vet.ucm.es

	References

Top Abstract Materials and Methods Results Discussion References

 

Abrams LS, Vitale S, and Jampel HD (1996) Comparison of three tonometers for measuring intraocular pressure in rabbits. Investig Ophthalmol Vis Sci 37: 940–944.[Abstract/Free Full Text]

Bailey SJ and Hourani SM(1995) Effects of suramin on-contractions of the guineapig vas deferens induced by analogues of adenosine 5'-triphosphate. Br J Pharmacol 114: 1125–1132.[Medline]

Bergmanson JPG (1982) Neural control of intraocular pressure. Am J Optom Physiol Opt 59: 94–98.[Medline]

Crosson CE and Gray T (1996) Characterization of ocular hypertension induced by adenosine agonists. Investig Ophthalmol Vis Sci 37: 1833–1839.[Abstract/Free Full Text]

Crosson CE and Petrovich M (1999) Contributions of adenosine receptor activation to the ocular actions of epinephrine. Investig Opthalmol Vis Sci 40: 2054–2061.[Abstract/Free Full Text]

Gomez-Villafuertes R, Gualix J, Miras-Portugal MT, and Pintor J (2000) Adenosine 5'-tetraphosphate (Ap₄), a new agonist on rat midbrain synaptic terminal P2 receptors. Neuropharmacology 39: 2381–2390.[CrossRef][Medline]

Greiner JV, Chanes LA, and Glonek T (1991) Comparison of phosphate metabolites of the ocular humors. Ophthalmic Res 23: 92–97.[Medline]

Gualix J, Abal M, Pintor J, and Miras-Portugal MT (1996) Presence of -adenosine tetraphosphate in chromaffin granules after transport of -ATP. FEBS Lett 391: 195–198.[CrossRef][Medline]

Hourani SM, Bailey SJ, Johnson CR, and Tennant JP (1998) Effects of adenosine 5'-triphosphate, uridine 5'-triphosphate, adenosine 5' tetraphosphate and diadenosine polyphosphates in guinea-pig taenia caeci and rat colon muscularis mucosae. Naunyn-Schmiedeberg's Arch Pharmacol 358: 464–473.[CrossRef][Medline]

Lee JW, Jeon SJ, Kong ID, and Jeong SW(1995a) Identification of adenosine 5'-tetraphosphate in rabbit platelets and its metabolism in blood. Kor J Physiol 29: 217–223.

Lee JW, Kong ID, Park KS, and Jeong SW (1995b) Effects of adenosine tetraphosphate (ATPP) on vascular tone in the isolated rat aorta. Yonsei Med J 36: 487–496.[Medline]

Lewis CJ, Gitterman DP, Schluter H, and Evans RJ(2000) Effects of diadenosine polyphosphates (ApnAs) and adenosine polyphospho guanosines (Ap_nGs) on rat mesenteric artery P2X receptors ion channels. Br J Pharmacol 129: 124–130.[CrossRef][Medline]

Lieberman I (1955) Identification of adenosine tetraphosphate from horse muscle. J Am Chem Soc 77: 3373–3375.[CrossRef]

Lobaton CD, Vallejo CG, Sillero A, and Sillero MA (1975) Diguanosine tetraphosphatase from rat liver: activity on diadenosine tetraphosphate and inhibition by adenosine tetraphosphate. Eur J Biochem 50: 495–501.[Medline]

Mateo J, Miras-Portugal MT, and Rotllan P (1997) Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells. Am J Physiol 273: C918–C927.

Maul E and Sears M (1979) ATP is released into the rabbit eye by antidromic stimulation of the trigeminal nerve. Investig Opthalmol Vis Sci 18: 256–262.[Abstract/Free Full Text]

Mitchell CH, Carre DA, MCGlinn AM, Stone RA and Civan MM (1998) A release mechanism for stored ATP in ocular ciliary epithelial cells. Proc Natl Acad Sci USA 95: 7174–7178.[Abstract/Free Full Text]

Peral A, Navas B, Peláez T, and Pintor J (2001) Regulación de la presión intraocular por nucleótidos de adenina. Arch Optom 5: 7–14.

Pintor J (1999) Purinergic signalling in the eye, in Nervous Control of the Eye (Burnstock G and Sillito A eds) pp 171–209, Harwood Academic Publishers, Amsterdam.

Pintor J, Díaz-Hernández MA, Gualix J, Gómez-Villafuertes R, Hernando F, and Miras-Portugal MT (2000a) Diadenosine polyphosphate receptors from rat and guinea-pig brain to human nervous system. Pharmacol Ther 87: 103–115.[CrossRef][Medline]

Pintor J and Peral A (2001) Therapeutic potential of nucleotides in the eye. Drug Dev Res 52: 190–195.[CrossRef]

Pintor J, Peral A, Hoyle CH, Redick C, Douglass J, Sims I, and Yerxa B (2002) Effect of diadenosine polyphosphates on tear secretion in New Zealand white rabbits. J Pharmacol Exp Ther 300: 291–297.[Abstract/Free Full Text]

Pintor J, Peral A, Navas B, Peláez T, and Gallar J (2000b) Effects of ATP and adenine nucleotides on intraocular pressure in New Zealand rabbits (Abstract). Investig Ophthalmol Vis Sci S255.

Pintor J, Peral A, Peláez T, Martín S, and Hoyle CHV (2003) Presence of diadenosine polyphosphates in the aqueous humor: their effect on intraocular pressure. J Pharmacol Exp Ther 304: 342–348.[Abstract/Free Full Text]

Rotllán P, Ramos A, Pintor J, Torres M, and Miras-Portugal MT (1991) Di(1,N 6-ethenoadenosine) 5', 5'''-P^1,P⁴-tetraphosphate, a fluorescent enzymatically active derivative of Ap₄A. FEBS Lett 280: 371–374.[CrossRef][Medline]

Small GD and Cooper C (1966) Studies on the occurrence and biosynthesis of adenosine tetraphosphate. Biochemistry 5: 26–33.[CrossRef][Medline]

Van Dyke K, Robinson R, Urquilla P, Smith D, Taylor M, and Trush M (1977) An analysis of nucleotides and catecholamines bovine medullary granules by anion exchange high pressure liquid chromatography and fluorescence. Evidence that most of the catecholamines in chromaffin granules are stored without associated ATP. Pharmacology 15: 377–391.[Medline]

Westhoff T, Jankowski J, Schmidt S, Luo J, Giebing G, Schlüter H, Tepel M, Zidek W, and van der Giet M (2003) Identification and characterization of adenosine 5'-tetraphosphate in human myocardial tissue. J Biol Chem 278: 17735–17740.[Abstract/Free Full Text]

Winkler H and Carmichael SW (1982) The chromaffin granule, in The Secretory Granule (Poisner AM and Trifaro JM eds) pp 3–78, Elsevier North Holland Biomedical Press, New York.

Yerxa BR (2001) Therapeutic use of nucleotides in respiratory and ophthalmic diseases. Drug Dev Res 52: 196–201.

Zimmermann H (1996) Extracellular purine metabolism. Drug Dev Res 39: 337–352.[CrossRef]

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